Isolation of Apigenin From Trigonella foenum Graecum Seed Extract Using Column Chromatograpy on Lab Scale

 

Ms. Shivani Watak, Dr. Mrs. Swati S. Patil

Prin. K.M. Kundnani College of Pharmacy, Cuffe Parade, Colaba, Mumbai.

*Corresponding Author E-mail: watak_shivani@rediffmail.com

 

 

 

1. INTRODUCTION :

Trigonella foenum-graecum is also employed as an herbal medicine in many parts of the world. Fenugreek has been reported to possess a curative gastric anti ulcer action, anti-bacterial, anti-helminthic, anti-fertility, carminative, tonic and aphrodisiac, anti- - diabetic activity and hypocholesterolaemic effects. Through our investigation on Trigonella foenum graecum was found that flavonoids Rutin and Apigenin are the major constituent of its methanolic extract. Moreove rthe literature point out that some activities can be especially related to these flavonoids such as antioxidant, anti-inflammatory, anticarcinogenic and antimutagenic. After performing TLC on methanolic extract of seed an attempt was made for separating  Apigenin from  column chromatograpy .The chromatographic separationwas performed by using Silica Gel for Column Chromatography (60-120# and 200-400#) and mobile phase comprising of butanol: ethylacetate (1: 1)  and flow rate 0.5ml/ min. The methods of determination of Apigenin include  HPTLC, UV-Vis spectrophotometry, IR spectroscopy and HPLC with standard Apigenin.

 

2. EXPERIMENTAL:

2.1. Plant Collection

Trigonella foenum graecum L. seeds were collected from the local market (APMC) Vashi, Navi Mumbai, Maharashtra in the month of May 2010.

 

Authentication of the collected material was carried out from Blatter Herbarium ST. Xavier’s College Mumbai by Dr. Rajendra D. Shinde, and its identity was confirmed to be Trigonella foenum graecum L, family Fabaceae, with hrrbarium accession no. 23844. Certificate of authentication is shown in fig. 1.

 

2.2. Extraction

Collected seeds were sundried for one day. These sundried seeds were further subjected to size reduction in the mixer grinder and then extracted. Extraction of Seeds of Trigonella foenum graecum was carried in soxhlet extractor using methanol as solvent. 10gm of dried and powdered seed were weighed and packed in filter paper, then extracted with methanol (300ml)at a temperature not exceeding 55ºc in a soxhlet apparatus. After completion of extraction the solvent was evaporated in evaporating dish on the water bath to obtain dry extract. The weight of dry extract was taken to find out extractive value

 

Table 1.1 Extractive value of Trigonella foenum graecum extract

Extract	Extractive value (in %w/w)
methanol	4.5

 

2.3. Requirements:

Plant material: Extract of Trigonella foenum graecum

Chemicals: n-butanol, Ethyl acetate, Methanol, Toulene (of Merck LR/AR Grade), Silica Gel for Column Chromatography (60-120# and 200-400#) (All solvents were distilled prior to use).

 

Apparatus: Column of different size, Test tubes of different size with taste tube stand,(Properly washed and dried), Sonicator, TLC[Silica gel G60 F₂₅₄ (Merck, Thickness 0.02mm) pre coated with Aluminium sheets] Plates, UV Chamber, Microwave Oven.

 

2.4. Methodology:

It consist of following steps (1,2)

1. Activation of Silica gel:

Silica gel (for Column chromatography 60-120 #, ) ~ 30g was taken in 100ml beaker and placed in microwave Oven at 50⁰c for 2 hours prior to use.

 

2. Sample Preparation:

500mg of Trigonella foenum graecum extract was taken in 5 ml of methanol and sonicate it up to 10 minutes and was properly covered with aluminium foil.

 

3. Slurry preparation (Wet Method):

Slurry of activated silica gel was prepared using n-butanol: ethylacetate (1:1) ratio.

 

4. Column Packing:

     A uniform well consolidated packing of the column is critical to the success of chromatographic separation.

     A clean dry column is aligned in a vertical position.

     A beaker is placed under the column outlet.

     The column was slowly and evenly filled about 2/3^rd fill with silica Gel slurry. The stop cock is opened to allow liquid to drain into the beaker powering the slurry down a glass rod held against the wall of the column will minimize bubbling and turbulence.

     The side of the chromatographic tube was gently tapped with a cork ring during the packing process, to make the silica gel compact.

     Meanwhile the stop cock is opened to allow the excess eluting solvent to run out. The bottom outlet of separation column is closed is very important, the column was not allowed to run dry.

 

5. Loading of column:

     After preparation of column, mobile phase was incorporated into the column and bottom outlet/stopcock is opened (1ml/min) for 10 minutes continuously.

     To allow the excess eluting solvent, run out and the mobile phase was drained   into beaker.

     After saturation of column, sample (5ml) was loaded into the same by dropper/volumetric pipette directly to the silica Gel layer from top of the column and covered it properly by aluminium foil.

     Allowed the mobile phase was drained continuously to the top of the column.

     The bottom outlet (stopcock) of column is opened to run with (1ml/min).

     The eluent flows down through the column.

     As the eluent passes down the column. The ‘separation Zones’ flow out of the column.

     Incorporation of mobile phase at some interval.

     Where the eluted material was collect into the taste tubes (2ml/taste tubes).

 

Figure 1.2 Image of column

 

6. Separation and Purification:

Each fractions collected in the test tube was spotted on a TLC plate. Then plate was developed using different mobile phase such as n-butanol:ethyl acetate (1: 1), toluene: ethyl acetate (2: 1), n-butanol:acetic acid: water(5: 1: 4).  The plate was observed for spot or spots to determine which compound is in each of the collected fractions. A light yellow colored fraction showed two spots in all the above mobile phases, but proper separation of two constituent observed in toluene : ethyl acetate,(2:1) hence it was used as final mobile phase for repeated column chromatography .thus purification of constituent was done by repeated CC by using  toluene : ethyl acetate, (2:1) as mobile phase. Compound isolated from column was collected, solvent was evaporated and weight of dried powdered drug was taken.

 

3. Characterisation of Isolated Compound By,^(3,4,5)

     Phytocemical analysis

     Co chromatography of isolated compound with standard Apigenin using HPTLC 

     Ultraviolet spectroscopy

     Infrared spectroscopy

     High performance liquid chromatography

 

3.1. Phytochemical Analysis

Table 1.2 Phytochemical analysis of isolated fraction

Extract Fraction Phytochemical Analysis
Colour	Pale yellowish green
Texture	Smooth, Powdery Material
Solubility	Soluble in ethanol, methanol,  and Water
Test for flavonoid Shinoda test	positive
TLC Spots	Single Spots,
yield	6.4mg from 500mg of extract

 

3.2. Co-chromatography of isolated compound with   

Standard apigenin using HPTLC

Reagents required

Standard Apigenin was procured from sigma Aldrich lab. Toluene, ethyl acetate, methanol (Merck LR/AR grade)

 

Preparation of sample and standard solution

100 µg/ml solution of isolated compound and standard Apigenin was prepared in methanol and applied on stationary phase.

 

Methodology

High performance thin layer chromatography (HPTLC) was performed on isolated compound from Trigonella foenum graecum extract and standard Apigenin. Both were subjected to qualitative evaluation using pre coated TLC silica gel G60 F254 (merck) plates as stationary phase. The plates were pre- washed in methanol for removing any impurities picked up by plate during storage in the laboratory environment. After pre- washing the plates were activated at 110-120º C for half an hour. Most suitable mobile phase to give better chemoprofile of the constituents. Linomat 5 applicator and Hamilton 100µl syringe was used for application. The chromatographic plate was scanned by using Desaga CD 60 densitometer and proquant software was used at 270nm for the purpose of obtaining a fingerprint of the isolated compound and standard Apigenin

 

Co- Chromatographic condition:

     Applicator:  Linomat 5 applicator and Hamilton 100 µL syringe

     Development Chamber: Camag double trough TLC development chamber

     Adsorbent : Silica gel 60 F₂₅₄ (pre-coated Aluminim plates)

     Chromatographic solvent: Toulene : ethyl acetate (2:1)

     Technique of development :      Ascending

     Length of run : 8cm

Visible:  yellowish green colored band.

Rf value of isolated compound : 0.69

Rf value of std Apigenin:            0.70

 

Track        1            2          3           4          5

 

Figure 1.3  Co- chromatography of isolated compound of Trigonella foenum graecum extract with std. Apigenin ( track 1-std Apigenin(20µl), track 2, 3,4,5 of isolated compound of concentration 5, 10, 20 and 40µl) were performed by using HPTLC at 270 nm present as follows

Figure  1.4   chromatogram showing overlay of std. Apigenin and isolated compound      (track 1-std Apigenin(20µl), track 2, 3,4,5 of isolated compound of concentration 5, 10, 20 and 40µl) were performed by using HPTLC at 270 nm present as follows

 

Figure 1.5 Representive chromatogram of standard Apigenin (20μl)

 

Figure 1.6 Representive chromatogram of   isolated compound (5μl)

 

Figure 1.7 Representive chromatogram of  isolated compound (10μl)

 

Figure 1.8 Representive chromatogram of isolated compound (20μl)

 

Figure  1.9 Representive chromatogram of  isolated compound (40μl)

 

Table 1.3 Analysis of co- chromatography

Component	Weight in mg	Application volume	Rf
Std Apigenin lane1	1mg/ml	20µl	0.70
Isolated lane 2	1mg/ml	5µl	0.69
Isolated lane 3	1mg/ml	10µl	0.69
Isolated lane 4	1mg/ml	20µl	0.69
Isolated lane 5	1mg/ml	40 µl	0.70

 

ULTRAVOILET SPECTROSCOPY

Instrument used: Jasco UV-550 UV/VIS spectrophotometer.

The isolated compound and standard Apigenin was dissolved in methanol to get a concentration of 10µg/ml. spectra measurement was done by scanning in the UV range from 200-600. The wavelength of maximum absorption (λ _max) was observed from the UV spectra

 

Figure  1.10 UV scan of standard Apigenin

 

Figure 1.11 UV scan of isolated compound of  Trigonella foenum graecum

 

INFRARED SPECTROSCOPY

IR spectrum of isolated compound and standard Apigenin was recorded using SHIMADZU IRAffinity-1. The isolated sample was examined by mixing the sample with KBr in the Ratio of 1:100 and applying the principals of diffuse reflectance spectral measurement. For IR spectra KBr was obtained from Merck industries

 

Figure  1.12  IR spectra of isolated compound from Trigonella foenum graecum

 

Figure 1.13  IR spectra of standard Apigenin

Table 1.4: Analysis of IR Spectra

	Standard Apigenin	Isolated compound
C-H stretching	3095.15	3028.24
O-H stretching	3327.2, 3502.13,3635.82	3641.60,3531.81
C= C stretching	1502.55	1500.62
C=O stretching	1649.16, 1608.54	1656.85 , 1604.77
-C-O streching	1354.03	1352.10
Benzene ring	1269.17, 1031.92	1222.87,1031.92

 

High Performance Liquid Chromatography:

Fingerprint analysis approach using high performance liquid chromatography (HPLC) has become the most potent tools for quality control of herbal medicines because of its simplicity and reliability. It can serve as a tool for identification, authentication and quality control of herbal drugs.

 

The chromatogram of isolated constituent and standard Apigenin was obtained by HPLC. The optimized Chromatographic conditions for HPLC analysis was as follows:

 

Preparation of standard solutions

10 mg of standard Apigenin was accurately weighed and was added to 10 ml HPLC grade methanol (Stock solution 1mg/ml). This stock solution was further diluted to get different concentration of 100μg/ml

The max of Apigenin in methanol was found to be 268nm. Hence the absorbance of the above mentioned standard solutions were recorded at 268nm.

 

Preparation of sample solution

10µg/ml solution of isolated fraction was prepared in HPLC grade methanol

 

Table 1.5  Chromatographic condition for HPLC

 

 

RESULT AND DISCUSSION

Weight of compound isolated from column chromatograpy was found to 6.4mg from 500mg of extract Co- chromatography of isolated compound with standard Apigenin showed Rf value for isolated compound and standard Apigenin was found to be same i.e. 0.70

 

The UV scan of standard Apigenin showed 3 peaks with λ- max value at 341nm, 269.5 nm and 227nm whereas isolated compound also showed 3 peaks with  λ- max values at 341nm, 270 nm and 223nm, which matches with the standard, hence both the compounds may be same

 

On comparing the IR spectra of isolated compound and standard Apigenin IR values for Benzene ring C-H, O-H, C=C, C=O. –C-O, stretching vibrations were similar. From the above values both the compounds may be same

 

The HPLC chromatogram of isolated compound depicted retention time at 6.37min at 272 nm which matched with standard Apigenin which showed retention time at 6.36 mins at 268nm indicates both are same.

 

Hence, evaluation of isolated compound by co-chromatography, UV, IR and HPLC indicates that compound isolated from column chromatography may be Apigenin.

 

 

Figure  1.14  HPLC chromatogram of standard apigenin

 

Figure 1.15  HPLC chromatogram of isolated compound

 

REFERENCES :

1      Dr. S.N.Meyyanathan, Isolation and purification of substance by column chromatography by department of pharmaceutical analysis, J.S.S.college of pharmacy, Tamilnadu, India. Pharma Info Net latest reviews (vol.2), 1/7/2004 –(07)

2        Dr G R Chatwal and sham k. Anand , instrumental methods of chemical Analysis  Publisher: Himalaya Pub. Ho, India. fifth enlarged ed. 2002 ,  2.647-2.655

3        Xiao-qing Chen, Jian-bo Xiao*RP-HPLC-DAD Detrmination of Flavonoids:  Separation of Quercetin, luteolin and Apigenin in Marchantia Convoluta iranina Journal of Pharmaceutical Research, 7 July 2005, artical 8, (Volume 4), Number 3, Page 175-181

4        Vijay Gokarn1,2*, Vidya Dighe1,Sasikumar Menon2, Bhalerao Khairnar1,2 online hplc-dad/uv-ms determination of major flavonoids rutin and isoquercitrin in two morus species. International Journal of Pharmaceutical Research and Development August - 2010 (Volume - 2 ) Issue – 6, Article No - 7  

5        Yuangang Zu,  Chunying Li, Yujie Fu and Chunjian Zhao  Determination of five flavonoids (catechin, CA; rutin, RU; quercetin, QU; kaempferol, KA; isorhamnetin, IS) in the extract of sea buckthorn (Hippophae rhamnoides L.) leaves. Journal of Pharmaceutical and Biomedical Analysis Volume 41, Issue 3, 7 June 2006, Pages 714-719

 

 

 

Received on 26.03.2012         Modified on 05.04.2012

Accepted on 30.04.2012         © AJRC All right reserved