Development of a New Isolation Technique and Validated RP-HPLC method for Quercetin and Kaempferol from Azadirachta indica leaves

 

Om Prakash1*, Debarshi Kar Mahapatra2, Ruchi Singh1, Namrata Singh1, Neelam Verma1 Akash Ved1

1Goel Institute of Pharmacy and Sciences, Faizabad Road, Lucknow, Uttar Pradesh, India

2Department of Pharmaceutical Chemistry, Dadasaheb Balpande College of Pharmacy, Nagpur 440037, Maharashtra, India

*Corresponding Author E-mail: opverma2007@gmail.com

 

ABSTRACT:

The aim of this study was to develop a new method for isolating the two major flavonoids, quercetin and kaempferol by using the RP-HPLC technique from Azadirachta indica leaves. The flavonoids were initially separated and identified by analytical column while isolated by the preparative column. The quercetin and kaempferol were identified by an external standard analytical RP-HPLC procedure. The fraction collection was set up through software and the peaks collected in the chromatogram were 7.42 min for quercetin and 11.39 min for kaempferol. The purity of the collected compounds showed UV area % purity of 86.67 and 89.23 for quercetin and kaempferol, respectively. The standard quercetin demonstrated λmax of 259 nm and 364 nm and the isolated quercetin displayed λmax of 256 nm and 365 nm, respectively. The standard kaempferol exhibited λmax of 267 nm and 368 nm whereas the isolated kaempferol presented λmax of 259 nm and 361 nm, respectively. Both the FT-IR spectra (standard and isolated) of quercetin and kaempferol superimpose with each other. The prominent spectral peaks were located at >3300 cm-1 (indicating –OH groups) and 1600-1700 cm-1 (representing the carbonyl function). The method has been found to be precise, accurate, linear, robust, economic, sensitive, and can rapidly isolate both the key phytoconstituents from the extracted natural materials. Thus, the research opens avenues for the application of this method in industry-based functions as well as quality control measures at varied operability scale.

 

KEYWORDS: Quercetin, Kaempferol, Azadirachta indica, RP-HPLC, Flavonoid, Phenolic.

 

 


INTRODUCTION:

Phenolic compounds are important secondary plant metabolites reported for diverse and significant biological and pharmacological activities like anti-bacterial, anti-fungal, anti-viral, anti-ulcer, anti-allergenic, and anti-inflammatory1-4.

 

These compounds also appear to be effective in certain kind of cancerous process and some cardiovascular diseases.5 Phenolic compounds are also used as a natural antioxidant or as a food additive to preserve food for organoleptic and nutritional quantities.6-7

 

Flavonoids are one of the important and largest classes of phenolic compounds, generally known to be present in plant and plant based product, associated with a broad spectrum of health-promoting effects8. The presence and concentration of these compounds vary even in the same plant at different ripening stages and organs of the same plant. Some plants are found to be an excellent source of phenolic compounds and suggested to use for preservation of food as well as to get health benefits.9 Quercetin and kaempferol (Figure 1) are the basic flavonoids possess anti-oxidant, anti-microbial, antiviral, anti-mutagenic, and anti-inflammatory properties10.

 

 

Figure 1. Structure of the phytoconstituents.

 

Azadirachta indica (A. indica) is known by Hindi name Neem, is a member of mahogany family Meliaceae.11-12 Different parts (roots, stem, bark, leaves, flower, fruits, and seeds) of the A. indica have been used traditionally for the treatment of infections, inflammation, fever, dental disorders and skin diseases13-15. More than 140 active compounds have been isolated from A. indica, including phenolic compounds, alkaloids, triterpenoids, flavonoids, carotenoids, ketones, and steroids.16-19

 

The aim of this study was to isolate the two major flavonoids, quercetin and kaempferol by using the RP-HPLC technique from A. indica leaves by establishing a new method and also to recognize the critical parameters essentially required for the identification. The flavonoids were initially separated and identified by analytical column while isolated by the preparative column.

 

MATERIAL AND METHODS:

Instrumentation:

The RP-HPLC system comprised of Shimadzu® LC-2010 CHT (Kyoto, Japan) with inbuilt SPD 20-AD UV-Vis detector. The separation was carried out by using reverse-phase column Intersil ODS-3 C-18 (5 µm, 4.6 mm×250 mm). The UV-spectrophotometric analysis was performed using the double-beam Shimadzu® Ultraviolet-Visible Spectrophotometer (Kyoto, Japan) of model UV-1800 which is equipped with a pair of 1 cm path length quartz cells. The weighing applications were carried out on Shimadzu® AUW220D balance. The pH was determined by utilizing Contech® digital pH meter. The sonication was done with a sonicator of Transonic Digital S model (USA).

 

Collection of plant:

The A. indica was collected from the local area of Lucknow, Uttar Pradesh, India. The fresh fully green leaves were selected at the initial stage of maturity during the month of January. The plant was rightfully authenticated from the Department of Pharmacognosy and submitted to the Institutional Museum for preservation. All the sample leaves were washed with plenty of water and dried in vacuum oven at 40°C for 48 hrs. The dry leaves were crushed with a commercial grinder and sieved by using 80-mesh. The powder sample was stored in the polyethylene bags in the desiccating cabinet for further analysis.

 

Preparation of extract:{Agrahar-Murugkar, 2005 #102}

20 g of dried leaves were macerated with 200 ml of distilled water. The aqueous extract was fractionated with 50 ml petroleum ether to remove the fatty materials. The aqueous extract was subsequently extracted with 50 ml of ethyl acetate. The ethyl acetate extract was dried over anhydrous H2SO4 and evaporated to dryness under N2 stream at 40°C.

 

Study and selection of wavelength:

The solutions of the standard phytoconstituents at a concentration of 10 μg/mL were scanned in the range of 400-200 nm in a 1 cm cell against blank sample using UV-Vis spectrophotometer in a triplicate manner to accurately measure the λmax and the mean was reported. The wavelengths of the spectrum chosen for the determination of the phytoconstituents were found to be 364 nm for quercetin and 368 nm for kaempferol. The most common wavelength employed for the detection was 366 nm.

 

Preparation of standard solution:

1 mg of both the standard phytoconstituents was transferred into separate 100 mL of volumetric and suitably diluted with acetonitrile and further sonicated to completely dissolve the substances. The final volume was made up to 10 μg/mL after cooling the solution to the room temperature.

 

Characterization of quercetin and kaempferol by RP-HPLC:

The two flavonoids were separated by analytical column Intersil ODS-3 C-18 (5 µm, 4.6 mm×250 mm) using mobile phase acetonitrile: methanol: aqueous solution of 0.2% formic acid in ratio of 60:10:30 at a flow rate of 1 ml/min in the total run time of 20 min at the isocratic mode by using UV-Vis detector.

 

Isolation of quercetin and kaempferol by RP-HPLC:

The obtained extract was sonicated for the duration of 20 min in sonicator and the sonicated extract was passed through the ultra membrane filter (pore size 0.45 μm) prior to injection in the sample loop. The obtained filtrate was thoroughly analyzed by HPLC system. The scaled preparative chromatographic procedure was similar to the analytical procedure, having the ability to collect the peak of interest. The quercetin and kaempferol were isolated and achieved by semi-preparative column Intersil ODS-3 C-18 (5 µm, 6 mm×250 mm), using mobile phase composition; acetonitrile: methanol: aqueous solution of 0.2% formic acid in a ratio of 60:10:30 at isocratic mode. The selection of the mobile phase was decisive and subsequently to numerous trials with the solvent mixtures, the critical parameters like degree of separation between the two components, the best possible symmetry of the obtained peaks, index of peak purity, and the number of theoretical plates covered were taken into account for choosing the best mobile phase. The acceptable preparative separation achieved using 20 μl injection. The flow rate was set of 10 ml/min.

 

Validation of method:

For estimating the linearity, different concentrations were selected at ranges of 25-150% of the target analyte concentrations. An equivalent amount of each solution was analyzed by chromatography and the obtained calibration graph (average area versus concentration) was plotted to determine the regression coefficient value (r2)20. The recovery was determined by spiking the phytoconstituents in placebo at a fixed concentration along with the placebo amount in the chromatography system. The % recovery, % mean, and % relative standard deviation (RSD) value were determined and expressed. 21 The precision of the method was studied by the inter-day and intra-day variability. The intra-day analysis was executed at three concentrations of the standard solution in a single day (intra-day) and on three-difference day (inter-day)22. The robustness of the method was studied and the effect of changing the flow rate by the value of +0.2 mL/min; i.e. 1.2 mL/min and wavelength by +2 nm (now 368 nm) while keeping the other chromatographic conditions stable23. All the experimental studies were done in a triplicate manner and the obtained mean was reported.

 

Spectrophotometric analysis:

The isolated phytoconstituents were dissolved in methanol and λmax was determined by UV-Vis spectrophotometer (Shimadzu® UV-1800, Japan) by scanning over the wavelength of 200 to 400 nm. The obtained peak (s) were compared with the standard quercetin and kaempferol. In addition to it, the Fourier-transformed Infrared Spectroscopy spectra (Shimadzu® IRAffinity-1, Japan) of the isolated phytoconstituents were determined using KBr method and compared with the standard.

 

Data analysis:

The retention time(s) and concentration(s) of phenolic acid were identified by using HPLC software (Cyberlab-Chrom HPLC).

 

 

RESULTS AND DISCUSSION:

Isolation of quercetin and kaempferol:

The previously reported literature revealed the utility of C8 and C18 columns of the dimension (250×4.5 mm i.d., 1.7 or 5 μm particle size) for the resolution of both of these phytoconstituents, therefore, in this study reverse-phase column Intersil ODS-3 C-18 (5 µm, 4.6 mm×250 mm) was employed. To accomplish the best separation and isolation of these two prime substituents, the composition, type, its pH, and the formulation of the mobile phase played a critical function. Numerous trials were taken using multiple solvent systems like water, methanol, acetonitrile, and various buffers of pH 3.0-6.0 and the selection was based exclusively on peak symmetry, theoretical plate, and peak purity index. An optimized pH of the mobile phase was maintained to prevent tailing and dissolution of silica of the column. The quercetin and kaempferol were identified by an external standard analytical RP-HPLC procedure where the standard characterization method represented characteristic peaks of quercetin and kaempferol (Figure 2). The fraction collection was set up through software and the peaks collected in the chromatogram were 7.42 min for quercetin and 11.39 min for kaempferol (Figure 3). The purity of the collected compounds showed UV area % purity of 86.67 and 89.23 for quercetin and kaempferol, respectively. The tailing factor (TF) of both the peaks averagely remained near to unity which expressed excellent peak symmetry and the asymmetric factor (AF) is nearly or equal to 1, as with the enhancement in the AF, the phenomenon of tailing becomes prominent. As per the analytical observations, TF value <2 represents the ideal Gaussian peak21.

 

Validation:

The linearity expression of both the phytochemical samples was found to be within the acceptable range. The regression coefficient values were more than 0.99 which indicated the desired level of linearity. The recovery of the two substances was determined using the calibration curve. The estimated % RSD values were noticed to be <2% in each case which described that the values were within the acceptable limit (Table 1). Thus, the newly developed analytical method for the estimation of quercetin and kaempferol has good accuracy. The precision data representing the intra-day and inter-day variability are described in Table 2. The % RSD values were noticed <2% in each case which depicted that the newly developed method was quite specific for the estimation of the phytoconstituents. The distinction between the intra-day and inter-day variability was perceived to be fairly nominal and also within the range. With the alteration in the chromatographic conditions, a very minute, but deliberate change was observed. The Rt values of quercetin and kaempferol shifted to 7.48 min and 11.46 min, respectively, when the flow rate was modified to 1.2 mL/min. Similarly, when the detection wavelength was modified by 2 nm, the observed Rt values were 7.46 min and 11.44 min, respectively, thereby indicating the robustness of the method.

 

 

Figure 2. RP-HPLC Chromatogram of Identification Quercetin and Kaempferol.

 

Figure 3. RP-HPLC Chromatogram of Isolation of Quercetin and Kaempferol.

 


 

Table 1. Recovery for accuracy studies.

Phytoconstituent

Content taken

Amount added to placebo

Amount recovered

% recovery

% mean

% RSD

Quercetin

10

10

19.86

99.3

99.93

1.11

10

10

20.19

100.95

10

10

19.91

99.55

10

15

25.17

100.68

99.88

1.17

10

15

24.85

99.4

10

15

24.89

99.56

10

20

30.11

100.36

99.87

1.04

10

20

29.90

99.66

10

20

29.88

99.6

Kaempferol

10

10

19.94

99.7

99.65

1.09

10

10

19.99

99.95

10

10

19.86

99.3

10

15

24.93

99.72

100.30

0.91

10

15

25.13

100.52

10

15

25.17

100.68

10

20

29.96

99.86

99.73

1.29

10

20

29.82

99.4

10

20

29.98

99.93

 

Table 2. Precision and repeatability of the phytoconstituents.

Phytoconstituent

Equivalent amount taken

Amount determined

(Inter-day)

% RSD

Amount determined (Intra-day)

% RSD

Quercetin

10

9.89

0.87

10.03

0.99

10

10.14

9.81

10

9.95

9.92

25

24.79

1.16

25.12

1.14

25

24.97

24.90

25

24.86

25.06

50

49.94

1.22

50.02

1.13

50

49.88

49.93

50

49.85

50.16

Kaempferol

10

9.92

0.82

9.95

1.01

10

9.96

10.07

10

9.90

9.99

25

24.93

1.11

24.99

1.18

25

24.89

24.97

25

24.81

24.85

50

49.91

1.19

49.80

1.07

50

49.84

50.05

50

49.98

49.91


Spectrophotometric analysis:

The standard quercetin demonstrated λmax of 259 nm and 364 nm and the isolated quercetin displayed λmax of 256 nm and 365 nm, respectively. The standard kaempferol exhibited λmax of 267 nm and 368 nm, whereas the isolated kaempferol presented λmax of 259 nm and 361 nm, respectively. These observations indicated the existence of flavonoid structure. The primary absorption originated from the π-π* transitions within the aromatic system (ring A) and the second absorption may be assigned to the transitions by the cinnamayl system (ring B). The second band appeared broader as a result of overlapping with the LMCT band. In addition to it, both the FT-IR spectra (standard and isolated) of quercetin and kaempferol superimpose with each other. The prominent spectral peaks were located at >3300 cm-1 (indicating –OH groups) and 1600-1700 cm-1 (representing the carbonyl function).

 

CONCLUSION:

The present research is a modernized version and commercially exploitable method for isolating the most important therapeutic natural products; quercetin and kaempferol from Azadirachta indica by utilizing RP-HPLC method. The method has been found to be precise, accurate, linear, robust, economic, sensitive, and can rapidly isolate both the key phytoconstituents from the extracted natural materials. Thus, the research opens avenues for the application of this method in industry-based functions as well as quality control measures at varied operability scale. Further research is needed for the still better chromatographic method for more rapid isolation and characterization of flavonoids from plant extracts.

 

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Received on 19.06.2018       Accepted on 05.09.2018     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2018; 8(3): 164-168.

DOI: 10.5958/2231-5675.2018.00030.3