A Simple Protocol for the Isolation of Quercetin from Trigonella foenum-graecum L. Leaves and Antioxidant analysis for Combating Toxicity

 

Durga. M¹*, Nathiya. S², Devasena. T³

¹Department of Biochemistry and Bioinformatics, Dr.MGR  Janaki College of Arts and Science, Chennai, Tamilnadu, India

²Department of Nanotechnology, Bharath Institute of Medical Sciences, Chennai.

³Centre for Nanoscience and Technology, Anna University,Chennai-600025, India

 

ABSTRACT:

Trigonella foenum-graecum L. is commonly known as fenugreek.  It is an annual herb, commonly used in many parts of India. In the present study total flavonoid estimation, antioxidant activity and isolation of quercetin were carried out. Sequential extraction was performed using chloroform, ethyl acetate and methanol. Highest yield was observed in methanol extract (10.04%). Among the three solvents ethyl acetate (3.2mg/g) showed maximum amount of total flavonoid content followed by chloroform (3.05mg/g) and methanol (1.0mg/g) extracts. Antioxidant potential was evaluated by DPPH free radical scavenging assay and Super oxide scavenging assay for ethyl acetate and chloroform extracts. Among the extracts ethyl acetate extract showed maximum antioxidant potential at 67.45% and 48.565% for DPPH free radical scavenging assay and Super oxide scavenging assay respectively. The isolation of quercetin was performed in ethyl acetate extract and validated using HPLC by comparing with known Standard quercetin.

 

 

 

1. INTRODUCTION:

Throughout history plants have been used by human beings for medicinal purposes and even in modern times have formed the basis for many pharmaceuticals products (1). The complex secondary metabolites produced by plants have established various therapeutic uses in medicine. The early history of modern medicine contains descriptions of phytochemicals, many of which are still in use (2). Huge scientific literature focuses on herbal extracts and their phytochemicals, encompassing hundreds and thousands of scientific papers, which has emerged over recent decades. Alkaloids, tannins, flavonoids and phenolic compounds are the most important bioactive constituents in treating various ailments (3).

 

Phenolics are ubiquitously found across the plant kingdom, with ~10,000 structures identified to date. Phenolics range from simple low-molecular weight compounds, such as the simple phenylpropanoids, coumarins, and benzoic acid derivatives, to more complex structures such as flavonoids, stilbenes, and tannins. Of these, the flavonoids represent the largest and most diverse group, comprising 6000 compounds, which share a common 6-carbon ring structure, with a 3-carbon bridge, which forms the 3rd ring. Flavonoids are categorized according to the modifications in the basic skeleton flavones, flavonols, flavanones, isoflavones, flavan-3-ols, and anthocyanins (4). Phenolics, and flavonoids in particular, are commonly present in plants and therefore represent an important component of a normal diet. Epidemiological studies have suggested associations between consumption of phenolic-rich foods or beverages and various deadly diseases, such as stroke, cardiovascular disease, and cancer (5) and neurologic disorders such as dementia AD (6,7).Phenols posses antioxidant and anticancer potential (8) and are recently considered as more potent antioxidants than Vitamin C, E and carotenoids (9). Phenols are also known to prevent aging and chronic heart disease (10). Trigonella foenum-graecum L. belongs to the family Papilionaceae and is commonly known as Fenugreek. It is an aromatic, 30-60 cm tall, annual herb, widely cultivated in India, Pakisthan, Egypt and Middle Eastern countries. The leaves are trifoliate and the leaflets are oblong-lanceolate and measure to 5 cm. Flowers are present in the leaf axils which measures to 12 – 18 mm long.

 

The fruits are almost straight and flattened with a pronounced beak; they are 50 – 110 mm long excluding the beak of 10 – 35 mm. Ten to twenty flat and hard, yellowish brown seeds are present in the curved seed pods. Seeds are angular- rhomboid, oblong or even cubic, and have a deep furrow dividing them into two unequal lobes. It is a highly aromatic plant which is used as a pot-herb, spice and fodder. It is widely grown in India and neighboring countries as a flavouring agent and fodder. Fenugreek is one of the oldest medicinal plants. It is rich in vitamins, calcium and β-carotene (11) it has been used for centuries for different female conditions, brain and nervous system ailments, skin, liver, hypercholesterolemia (12) and metabolic disorders. It is also considered highly beneficial for respiratory and gastrointestinal problems (13).It is a highly potent herb for females, since it helps relaxing the uterus and relieving menstrual pains, and is an excellent stimulator of milk production in nursing mothers (14).Fenugreek is also reported for antimicrobial, anticancer, antiviral, antitumor, antimicrobial, and anti inflammatory properties (15).

 

The present study involves the investigation of phyto-constituents and antioxidant property of Trigonella foenum-graecum L. leaf extracts. In this study a three solvent sequential extraction procedure was adopted for the isolation of Quercetin. The study also attempts in the purification and validation of quercetin using column chromatography, Preparative Thin Layer Chromatography (PTLC) and High Pressure Liquid Chromatography (HPLC) methods.

 

2. MATERIALS AND METHODS:

2.1 Plant Collection:

The leaf materials of Trigonella foenum-graecum L. were collected from field near Thiruvallur District, Tamil Nadu, India. It was authenticated by Professor, Dr. Jayaraman. The leaves were separated from other parts, washed, cleaned and shade dried for further use.

 

2.2 Preparation of Plant extract:

The plant materials (leaves of Trigonella foenum-graecum L.) were shade-dried at room temperature for two weeks, after which it was ground to a uniform powder. The extracts of the leaf samples were prepared in a sequential procedure by soaking 100 g of dried powder in 900 ml each of different solvents (ethyl acetate, chloroform and methanol) for 48 h. At the end of each successive extraction, the extracts were filtered using Whatman filter paper. The filtrate was concentrated under reduced pressure in vacuum at 40°C for 25 min using a rotary evaporator (Superfit-Rotavap, India). The percentage yield of extracts was calculated.

 

2.3 Phytochemical Screening:

Phytochemical screening for major constituents was undertaken using standard qualitative methods. Screening test were performed for Carbohydrates, Tannins, Saponins Flavonoids, Cardiac glycosides, Terpenoids, Triterpenoids, Alkaloids, Quinones, Phenols, Coumarins, Glycosides, Proteins, Steroids and phytosteroids (16,17).

 

2.4 Estimation of Total Flavonoid Content:

Total flavonoid content (TFC) in the leaf extracts (Chloroform, ethyl acetate and methanol extract) was determined using the method described by (18).The flavonoid content was determined by aluminium chloride method using Quercetin as standard. Extracts and Standard were prepared in ethanol (1 mg/ml). 0.1ml of extract was mixed with 0.9ml of distilled water in test tubes, followed by addition of 75μL of 5% sodium nitrite solution. After six minutes, 150μL of a 10% aluminium chloride solution was added and the mixture was allowed to stand for further five minutes after which 0.5 ml of 1M sodium hydroxide was added to the reaction mixture. The reaction mixture was brought to 2.5 ml with distilled water and mixed well. The absorbance (A) was measured immediately at 510 nm using a spectrophotometer (Elico, India). Assay was performed in triplicates. A calibration curve was generated using various concentrations of Quercetin (20 - 140µg). Blank was maintained without extract or Quercetin and substituted with 0.1 ml of ethanol. Results were expressed as mg of Quercetin equivalent/g of dry weight (mg QE/g) of extracts. Total flavonoid content in the plant extract was calculated using the formula:

                                         QE x V

Total flavonoid content = -------------

                                                    m

 

Where QE, is the Quercetin equivalence (mg/ml) or concentration of Quercetin solution established from the calibration curve; V, is the volume of extract (ml) and m, is the weight of the pure plant extract (g).

 

2.5 Antioxidant activity:

2.5.1 DPPH Free Radical Scavenging Activity:

The ability of Ethyl acetate and chloroform extract of Trigonella foenum - graecum L. to annihilate the DPPH radical (1,1-diphenil-2-picrylhydrazyl) was investigated by the method described by (19). Stock solution of leaf extracts was prepared to the concentration of 1mg/ml. Different concentrations (50, 100 and 150µg) of each extracts were added, at an equal volume, to methanolic solution of DPPH (0.1%). The reaction mixture is incubated for 30 min at room temperature; the absorbance was recorded at 517 nm. The experiment was repeated three times. BHT (Butylatedhydroxytoluene) was used as standard control. IC₅₀ values were calculated, which denote the concentration of sample, which is required to scavenge 50% of DPPH free radicals. The annihilation activity of free radicals was calculated as percentage (%) inhibition according to the following formula:

                          A of control – A of Test

% of Inhibition = --------------------------     X100

                               A of control  

 

2.5.2 Superoxide anion scavenging activity:

Measurement of superoxide radical scavenging activity was performed by the method described by (20). The superoxide anions generated by phenazinmethosulfate (PMN)/ nicotinamid-adenin-dinucleotidphosphate, reduced form NADPH system, were detected by the reaction with nitro blue tetrazolium (NBT). Stock solution of leaf extracts and Quercetin (standard) was prepared to the concentration of 1mg/ml. The reaction mixture contained 1ml of Nitro blue tetrazolium (NBT) solution (312µM prepared in phosphate buffer, pH-7.4), 1ml of Nicotinamide adenine dinucleotide (NAD) solution (936 µM prepared in phosphate buffer, pH-7.4) and samples at different concentration (50, 100 and 150µg) or standard solution (50, 100 and 150µg) obtained from stock solution. These were added and finally the reaction was accelerated by adding 100µl phenazinemethosulfate (PMS) solution (120 µM prepared in phosphate buffer, pH-7.4). The reaction was incubated at 25°C for 5 minutes and absorbance was measured at 560nm against the corresponding blank solutions. Blank consists of all the reagents, except for the extract or standard solution which is substituted by water. The annihilation activity was calculated as percentage of inhibition according to the following formula:

          Absorbance of Control - Absorbance of sample

% of Inhibition = ------------------------------------ X 100

                            Absorbance of Control

 

2.6 Column Chromatography:

The admixture was prepared with ten grams of the crude ethyl acetate extract and packed in a silica gel column (100–200 mesh, 100 g). This was followed by elution with 100% hexane, ethyl acetate and methanol in the ratio of 75:25, 50:50, 25:75 and 100% methanol. Based on the thin layer chromatogram (TLC) profile, the elutants were pooled into five fractions.

 

2.7 Isolation of Quercetin:

The eluted fractions were subjected for quercetin determination with reference compound quercetin through preparative thin layer chromatography( PTLC) method and High pressure liquid chromatography (HPLC) method (21).  Glass plates (20 x 20 cm) thickly coated (0.4-0.5 nm) with silica gel 'G' (45 gm/80 ml water) and the coated plates were activated at 100°C for 30 minutes and cooled at room temperature. To this the column chromatography elutants were applied on plates and the developed plates were air dried and visualized under ultra violet (UV) light. Each of the fluorescent spots coinciding with standard quercetin was marked. The marked spots were scrapped and collected separately and eluted with ethanol. The elutant was crystallized with chloroform and subjected to HPLC studies (Water associates, column-microporasil, 80% hexane and 20% ethyl acetate, chart spectra 1 cm/min, 0.5 ml/min UV detector at 254 nm).

 

2.8 Statistical Analysis:

The experimental data are expressed as Mean ± Standard Error. The data were subjected for Subjected for One way ANNOVA (SPSS 16.0) and the significance difference between the samples was calculated using Turkeys test. Results were considered to be statistically significant. IC₅₀ value is calculated using PROBIT (SPSS 16.0).

 

3. RESULTS:

3.1 Extraction and Phytochemical Screening:

Plant materials were collected and processed for sequential extraction using ethyl acetate, chloroform and methanol. The extractive yields are tabulated (Table 1) for Trigonella foenum-graecum L. The Preliminary phytochemical results in table 2 reveals the presence of Flavonoids, Phenols, Alkaloids, carbohydrates, triterpenoids and coumarins in all the three solvent extracts of Trigonella foenum-graecum L. quinones are present in methanol extracts. Tannins, steroids, phytosteroids and glyccosides were found to be absent.

 

Table 1: Extractive value of Trigonella foenum-graecum L. Leaf powder

Solvent	Percentage W/W
Ethyl acetate	2.5
Chloroform	3.88
Methanol	10.04

 

 

Table 2: Phytochemical screening - Leaf extracts of Trigonella foenum–graecumL.

Test	Ethylacetate	Chloroform	Methanol
Carbohydrate	+	+	+
Tannins	-	-	-
Saponin	-	-	-
Flavonoid	+	+	+
Alkaloid	+	+	+
Quinones	-	-	+
Glycosides	-	-	-
Cardiac glycosides	-	-	-
Terpenoids	-	-	-
Triterpenoids	+	+	+
Phenols	+	+	+
Coumarins	+	+	+
Steroids and Phytosteroids	-	-	-

“+” indicates presence and “-” indicates absence of phytochemical

 

3.2 Estimation of Total Flavonoid:

The total flavonoid content of Trigonella foenum- graecum L. leaf extracts were measured by Aluminium chloride reagents in terms of Quercetin equivalent (QE) and the results were tabulated in Table 3. The results indicate that ethyl acetate extract of Trigonella foenum- graecum L. possess high flavonoidcontent when compared to other solvent extracts. Total flavonoid content of samples (Fig 1) was obtained in comparison with the Quercetin standard.

 

3.3 DPPH Radical Scavenging Activity:

DPPH radical reacts with suitable reducing agents losing colour with the number of electrons consumed, which was measured spectrophotometrically at 517 nm. The Radical scavenging activity of Trigonella foenum–graecum L. is represented in Fig 2. Scavenging potential of the extracts was increased with increase in concentration of the sample. Among the various solvent extracts tested ethyl acetate extract showed maximum activity, when compared to chloroform extract. Scavenging potential of the samples in comparison to the BHT standard is described in Table 4.

 

Table 3: Estimation of Total Flavonoid content of Trigonella foenum–graecumL.

Solvent	A at 415nm	Total flavonoid content (mg/g)	Mean±S.E
Ethyl acetate	0.607	3.2	0.150
Chloroform	0.677	3.05	0.030
Methanol	0.299	1.0	0.025

 

 

Table 4: DPPH radical scavenging activity of Trigonella foenum–graecum L.

 

Table 5: Superoxide anion scavenging activity of Trigonella foenum–graecum L.

 

 

Table 6: Percentage yield of column chromatography fractions

Fraction No.	Solvent	Yield (g)
1	Hexane – 100%	0. 89
2	Ethyl acetate – 100%	4.2
3	Ethyl acetate : Methanol – 75:25%	1.8
4	Ethyl acetate : Methanol – 50:50%	1.35
5	Ethyl acetate : Methanol – 25:75%	0.650
6	Methanol – 100%	0.510

 

3.4 Superoxide Scavenging Activity:

The capacity to annihilate superoxide anions depended on the extraction medium. Chloroform extract showed lower scavenging activity against superoxide anion than ethyl acetate extract (Fig 3). The scavenging potential of different extract was compared with the standard Quercetin and the results are tabulated in table 5.

 

 

3.5 Column Chromatography:

Fractioning of ethyl acetate extract was performed using column chromatography. The solvent profile and obtained yield of various fractions are indicated in table 6. The detection of Quercetin was confirmed by subjecting the elutants through TLC (Fig 4).

 

3.6 Isolation of Quercetin:

The plates developed under UV light showed fluorescent spots in 100% ethyl acetate fraction similar to standard quercetin compound. The elutant was subjected to HPLC, retention time of sample and standard were compared (fig 4 and fig 5), which confirms the detection and isolation of quercetin from the leaf extracts of Trigonella foenum – graecum L.

 

 

Figure 4: HPLC analysis of standard Quercetin

 

Figure 5: HPLC analysis of fraction

 

4. DISCUSSION:

Medicinal values of the plants are based on the phyto-constituents of the plant. Flavonoids, alkaloids, phenols, saponins and tannins are considered as major bioactive compounds. Through phytochemical prospecting of the extracts, it was possible to determine the presence of diverse classes of secondary metabolites that shows a wide variety of biological activities. Flavonoids are available in wide range of medicinal plants, herbs and food source. These flavonoids have remarkable pharmacological property. Total flavonoid content of Trigonella foenum–graecum L. was measured by aluminium chloride colorimetric method which measures the complex formed by the sample with aluminum chloride (22). The assay reveals that total flavonoid content of 3.2mg/g (Mean±S.E: 0.1150) in ethyl acetate extract was higher when compared to 3.05mg/g (Mean±S.E: 0.030) and 1.0mg/g (Mean±S.E: 0.025) of total flavonoid content from chloroform and methanol extract respectively.

 

Free radicals are well known reactive molecules mainly derived from univalent reduction of oxygen. Scavenging and diminishing the formation of oxygen‐derived species are not 100% efficient. Micro nutrients or antioxidants taken as supplements are particularly important in diminishing the cumulative oxidative damages (23). DPPH assay is one of the oldest indirect method for determining the antioxidant potential, which is based on the ability of the stable free radical 2, 2-diphenyl-1-picrylhydrazyl to react with hydrogen donors. DPPH shows a strong absorption band at 517 nm in visible spectrum (deep violet colour). The bleaching ability of DPPH in the test samples indicates the capacity of the test drugs to scavenge free radicals (24). The assay emphasis antioxidant potential of ethyl acetate extract was 67.45% (p = 0.003). The inhibition potential was relatively higher in methanol extract of Trigonella foenum – graecum L. when compared to 42.79% (p = 0.002) in Ethyl acetate extract. The scavenging activities of crude extracts are found to be more significant when compared with control (p ≤ 0.005). The IC₅₀ value was obtained at 62.05µg and 175.29µg for ethyl acetate and chloroform extracts of Trigonella foenum – graecum L.

 

The superoxide anion radicals are derived in PMS‐NADH‐NBT system, where the decrease in absorbance at 560nm with antioxidants (25) indicates the consumption of superoxide anion in the reaction mixture, thereby exhibiting a dose dependent increase in superoxide scavenging activity. The assay reveals that antioxidant potential of methanol extract was 48.56% (p=0.001). The scavenging potential was relatively higher in ethyl acetate extract when compared to 44.95% (p=0.000) of chloroform extract. Superoxide anion scavenging of leaf extracts are found to be significant when compared with control (p ≤ 0.005). IC50 value was observed at 156.273µg for ethyl acetate extract and 166.508µg for chloroform extract of Trigonella foenum – graecum L.

 

The scavenging potential of the extracts were compared with a known reducing chemical BHT and quercetin. The activity of Trigonella foenum – graecum L. crude leaf extracts was comparatively lower than the known standards. The phytochemical compounds are known to support bioactivity of medicinal plants and thus responsible for the antioxidant activities of this plant extract. Flavonoids have been shown to exhibit their actions through effects on membrane permeability, and by inhibition of membrane-bound enzymes such as the ATPase and phospholipase A2 (26), and this property may explain the mechanisms of antioxidative action of Trigonella foenum – graecum L. The HPLC analysis of the elutant fractionated by column chromatography revealed the presence of Quercetin in the leaf extracts of Trigonella foenum – graecum L.

 

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