HPTLC Phytochemical Profiling and Simultaneous Quantification of Quercetin and Gallic acid in Neolamarckia cadamba (Roxb.)

 

Anant Kumar Srivastav1*, Shikhar Verma2, Himani Awasthi1, Santosh Kumar1

1Hygia Institute of Pharmaceutical Education and Research,

Ghaila Road, Balrampur, Lucknow, Uttar Pradesh 226020, India.

2Maharishi School of Pharmaceutical Sciences, Maharishi University of Information Technology (MUIT),

IIM Road, Indrapuri Colony, Diguria, Aziz Nagar, Lucknow, Uttar Pradesh 226013.

*Corresponding Author E-mail: anantsrivastava88@gmail.com, shikhar.verma@muit.in, himani1july@gmail.com, santosh@hygia.in

 

ABSTRACT:

HPTLC analysis of Neolamarckia cadamba (Roxb.) Bosser commonly known as kadamb reveals presence of several bioactive compounds along with percentage of quercetin and gallic acid in the methanolic extract of test samples. Quantification of markers (quercetin and gallic acid) along with phytochemical profiling of Neolamarckia cadamba was done at 254nm and 366nm wavelength through HPTLC CAMAG scanner III. Quantification was done by using peak area and peak height. Chromatograms and tracks obtained reveals that the test samples, sample 1 and sample 2 have quercetin 1.29% and 0.77% respectively. Phytochemical profiling of sample 1 and sample 2 reveals presence of 16 and 10 unknown bioactive compounds respectively denoted by the number of peaks present in chromatograms. As this study acknowledging phytochemical profiling, quantification of quercetin and gallic acid will be accountable for further research towards pharmacognostic and therapeutic value of Neolamarckia cadamba.

 

KEYWORDS: Neolamarckia cadamba, HPTLC, Chromatogram, Quercetin, Gallic acid.

 

 


INTRODUCTION:

Traditional drugs from medicinal plants have key barrier that they do not have quality measures and documentation to produce their role in treatment of various diseases and this requires standardization of practices to assure their qualities and justify its safety and efficacy.1-3 Neolamarckia cadamba (F: Rubiaceae) is a very important plant in the management of several diseases like eyes infections, skin diseases, stomach disorders, gum related problems, blood related issues and urinary disorders.4 The ethnomedicinal application and nutritive value of N. cadamba reveals that its bark have antioxidant, antigenotoxic and cytotoxic activity while its nutritious fruits have antioxidant, anthelmintic, membrane stabilizing and glucose level controlling properties.

 

The immune modulation activity of leaf extract utilized against cervical cancer also studied.5-9 Apart from above studies ethanolic extracts of dried leaves of Neolamarckia cadamba is also used in inflammation, anti-microbial activity, anti-fungal activity, wound healing activities. However a very few plant chemicals like sapogenins: cadambagenic acid, quinovic acid, and beta-sitosterol from bark, alkaloids like cadambine, 3 alpha-dihydrocadambine and isohydrocadambine have been reported.10-15

 

There are many techniques available for screening of plants products16-19 in which HPTLC is one of the which is worldwide accepted and very fast and inexpensive.20-24 Present study is designed to investigate the phytochemical profiling of aerial parts of the N. cadamba along with quantification of quercetin and gallic acid.

 

MATERIALS AND METHODS:

Collection, identification, and authentication of plant:

The aerial parts of Neolamarckia cadamba (Roxb.) Bosser syn. Anthocephalus cadamba (Roxb.) Miq. commonly known as kadamb was collected in the month of July 2022 from faizullaganj, ghaila road, Lucknow. The identification has been done on the basis of macroscopic studies of the sample followed by in-depth scrutiny of literature and identical the sample with authentic samples deposited in the RHMD (Raw Material Herbarium and Museum, Delhi). The plant authentication was done from CSIR-NIScPR under authentication no. NIScPR-RHMD/Consult/2022/4173-74-1.

 

Extraction:

The plant materials were washed, shed dried, powdered and subjected to successive extraction initially from n-hexane (69oC). After completion of 10 cycles of continuous extraction thimble was removed and powder was air dried. Again this dried powder was extracted with hydroalcohol (1:1, v/v) till at least 10 cycles completed. To obtain dry product excess solvent evaporated by vacuum rotary evaporator. The dried extract was stored in air tight container till the experiments were carried out.

 

Preparation of standard solution:

0.5mg/ml of quercetin and gallic acid prepared with HPTLC grade methanol were prepared.

 

Preparation of plant samples:

Test samples were prepared by dissolving 10grams of dried extract into 1ml of HPTLC grade methanol and filtered with 0.22m filter.

 

Instrumentation:

CAMAG TLC scanner III, winCATS software and LINOMAT 5 auto sprayer fitted with nitrogen cylinder and 100l syringe IN CAMAG HPTLC system were used.

 

Chemicals and Solvents:

All chemicals and solvents were of analytical and chromatographic grade. Reference markers were gifted by the National Botanical Research Institute (NBRI), Lucknow.

 

Chromatographic Conditions:

No pre washing but manual modification of plate (E-Merck KGaA) (20x10cm) pre coated with 0.2mm silica gel was used. Activation of plate at 40oC before analysis was done. Test samples were applied in 10l and 20l, 5mm above from bottom of the plate at constant rate 150nl/s. Mobile phase consisted of toluene: ethyl acetate: formic acid in 6:4:0.3 (v/v/v) were used in the developing chamber and after 5 minutes scanning at short 254nm and long 366nm wavelength through CAMAG scanner III was performed (Table 1).

 

UV active compounds present on TLC plate will appear as dark spot on a bright background due to fluorescence quenching at 254nm while at 366 nm appears as bright spot on a dark background11.

 

Table: 1 Parameters used for HPTLC

Parameters

Values

Calibration Parameter

Calibration mode

Multiple level

Statistics mode

CV

Evaluation mode

Peak height and peak area

Linomat 5 application parameters

Spray gas

Nitrogen

Sample solvent type

Methanol

Dosage Speed

150nl/s

Predosage volume

6l

Syringe size

100l

Application position

5.0mm

Band length

6mm

Solvent front position

98.0mm

Detection CAMAG TLC scanner

Number of track

4

Position of track

5.0mm

Distance between track

9.4mm

Scan start position Y

5.0mm

Scan end position Y

98.0mm

Slit dimension

6.00 x 0.30mm, micro

Optimize optical system

Light

Scanning speed

20mm/sec

Data resolution

100m/step

Integration: properties

Baseline correction

Lowest slope

Peak threshold min. slope

5

Peak threshold min. height

10AU

Peak threshold min. area

50

Peak threshold max. height

990AU

Track start position

5.0mm

Track end position

98.0mm

Display scaling

Automatic

Measurement

Wavelength

254nm and 366nm

Lamp

D2/Hg

Measurement type

Remission

Measurement mode

Absorption/fluorescence

Optical fibre

Second order/K400

Detector mode

Automatic

PM high voltage

181V

 

Calibration curve of quercetin and gallic acid:

Standard concentration ranging from 2 to 10g/spot prepared from 0.5mg/ml stock solution applied in 4l, 8l, 12l, 16l, 20l on HPTLC plate to obtained standards 2g, 4g, 6g, 8g, 10g/spot respectively. Calibration curves were obtained by plotting absorbance unit against concentration of standards (Fig. 6 and Fig. 7).

 

RESULTS AND DISCUSSION:

HPTLC chromatograms of test samples reveal that the phytochemical profiling and quantification of quercetin and gallic acid as illustrated in the figures and tables below. The chromatograms were obtained at short (254nm) (Fig. 1) and long (366nm) UV wavelength (Fig. 2). The Rf values, peak area, peak height and percentage area of unknown bioactive compounds depicted in tables 2, 3, 4, 5. Images of the TLC plate were taken at 254nm and 366nm represents their tracks for standard quercetin (track 1), gallic acid (track 2) and test samples (track 3 and track 4).

 

The HPTLC chromatogram obtained by standard quercetin (fig. 3) and gallic acid (fig. 4) showing their Rf values for quercetin at Rf 0.50 in track 1 and gallic acid at Rf 0.29 in track 2.

 

Chromatograms representing standard quercetin and gallic acid showed more than one peak representing that there were few impurities along with standards.

 

There were two test samples (sample 1 and sample 2). Sample 1 was initially extracted with n-hexane while sample 2 with hydro-alcoholic solvent. Calibration curve of standard quercetin and gallic acid was shown in fig. 6 and fig. 7 respectively. Chromatogram of sample 1 and sample 2 represents 16 peaks and 10 peaks respectively representing number of bioactive compounds bioactive compounds. The number of peaks denotes the number of bioactive compounds in the samples.

 

Sample 1 and sample 2 chromatogram represents peak at Rf 0.52 (track 3 and track 4) when compared to standard quercetin chromatogram quercetin peak Rf 0.50 ( track 1) was found that quercetin was present in sample 1 and sample 2 as there were approximately similar Rf values (0.50) of the compound when compared to standard quercetin Rf value (0.50).

 

Chromatogram of sample 1 and sample 2 when compared with standard gallic acid reveals that no Rf value of standard gallic acid is matching with sample Rf value denoting absence of gallic acid in both samples. Thus study concludes quercetin was detected in sample 1 and sample 2 while gallic acid was not detected in both samples. Quantification was done by using evaluation parameters peak area and peak height and quercetin was found to be 1.29% in sample 1 and 0.77% in sample 2.

 

Fig 1: Image of TLC Plate at 254 nm

 

Fig 2: Image of TLC Plate at 366 nm

 

Fig. 3: HPTLC Chromatogram of standard quercetin

 

Fig. 4: HPTLC Chromatogram of standard gallic acid

 

Fig. 5: HPTLC Chromatogram of standard Q and GA

 

 

Fig. 6: Calibration curve of quercetin

 

 

Fig. 7: Calibration curve of GA

 

 

Fig. 8: HPTLC Chromatogram of sample 1

 



Fig. 9: HPTLC Chromatogram of sample 2


 

Table 2 or Track 1: HPTLC peak table of standard quercetin

Track 1, ID: Standard 1

Peak

Start Position (Rf)

Start Height

(AU)

Max. Position

(Rf)

Max. Height

(AU)

Max %

End Position

(Rf)

End Height

(AU)

Area

(AU)

Area %

Assigned substance

 

1.

-0.02

0.5

-0.01

33.5

5.52

0.02

0.1

628.8

3.75

Unknown

 

2.

0.43

25.9

0.48

294.5

48.59

0.50

22.5

7340.2

43.81

Q

 

3.

0.51

22.9

0.53

34.9

5.76

0.55

19.2

991.6

5.92

Unknown

 

4.

0.63

23.4

0.64

27.1

4.47

0.66

22.0

618.6

3.69

Unknown

 

5.

0.78

33.4

0.86

89.8

14.82

0.86

36.8

4713.5

28.13

Unknown

 

6.

0.86

86.9

0.87

90.9

15.00

0.90

0.7

2086.8

12.46

Unknown

 

7.

0.95

0.4

0.96

23.6

3.90

0.97

5.8

229.9

1.37

Unknown

 

8.

0.97

6.2

0.98

11.7

1.94

0.99

0.0

144.9

0.87

Unknown

 

 

 

Table 3 or Track 2: HPTLC peak table of standard Gallic acid

Track 2, ID: Standard 2

Peak

Start Position (Rf)

Start Height

(AU)

Max. Position

(Rf)

Max. Height

(AU)

Max %

End Position

(Rf)

End Height

(AU)

Area

(AU)

Area %

Assigned substance

1.

-0.02

0.5

-0.01

38.0

3.86

0.01

3.2

471.0

1.60

Unknown

2.

0.21

18.5

0.25

499.5

50.76

0.29

20.0

16358.1

55.64

GA

3.

0.42

29.7

0.45

176.0

17.88

0.48

26.0

3926.3

13.36

Unknown

4.

0.49

29.9

0.51

45.5

4.62

0.53

35.6

1245.5

4.24

Unknown

5.

0.74

36.1

0.77

48.4

4.92

0.77

46.2

1536.1

5.23

Unknown

6.

0.79

53.7

0.83

87.8

8.92

0.84

34.4

3525.3

11.99

Unknown

7.

0.86

84.9

0.87

88.9

9.03

0.89

35.4

2335.1

7.94

Unknown

 

Table 4 or Track 3: HPTLC Peak table of sample 1

Table 4, Track 3 ID: HPTLC Peak table of sample 1

Peak

Start Position (Rf)

Start Height

(AU)

Max. Position

(Rf)

Max. Height

(AU)

Max %

End Position

(Rf)

End Height

(AU)

Area

(AU)

Area %

Assigned substance

1.     

-0.03

1.0

-0.01

131.5

7.51

0.01

27.8

1725.6

2.83

Unknown

2.     

0.01

28.1

0.01

30.0

1.71

0.03

10.3

579.4

0.95

Unknown

3.     

0.03

10.5

0.06

41.8

2.39

0.08

1.3

816.4

1.34

Unknown

4.     

0.09

1.3

0.12

43.0

2.46

0.16

21.7

1604.5

2.63

Unknown

5.     

0.22

4.7

0.23

13.8

0.79

0.24

5.4

199.1

0.33

Unknown

6.     

0.53

8.1

0.36

18.5

1.06

0.36

16.0

362.2

0.59

Unknown

7.     

0.37

15.7

0.43

90.6

5.18

0.43

37.1

3118.3

5.11

Unknown

8.     

0.44

87.4

0.45

93.6

5.34

0.46

32.4

1956.3

3.20

Unknown

9.     

0.47

90.3

0.50

182.8

10.44

0.52

35.3

5428.7

6.89

Q

10.   

0.52

65.4

0.53

87.0

4.97

0.55

36.0

1939.8

3.26

Unknown

11.   

0.55

67.1

0.56

77.9

4.45

0.58

56.7

1969.2

3.23

Unknown

12.   

0.59

55.9

0.67

111.3

6.35

0.71

34.5

9224.2

15.11

Unknown

13.   

0.71

64.7

0.76

343.1

19.60

0.79

47.0

14704.3

24.09

Unknown

14.   

0.79

147.4

0.80

161.9

9.24

0.81

55.6

3284.2

5.38

Unknown

15.   

0.81

156.2

0.84

288.2

16.46

0.88

28.1

13142.3

21.53

Unknown

16.   

0.95

30.3

0.97

36.2

2.07

0.98

30.3

937.4

1.54

Unknown

 

Table 5 or Track 4 ID: HPTLC Peak table of sample 2

Table 5, Track 4 ID: HPTLC Peak table of sample 2

Peak

Start Position (Rf)

Start Height

(AU)

Max. Position

(Rf)

Max. Height

(AU)

Max %

End Position

(Rf)

End Height

(AU)

Area

(AU)

Area %

Assigned substance

 

1.

-0.03

5.2

-0.01

695.8

69.36

0.03

43.2

13623.1

50.87

Unknown

 

2.

0.03

44.1

0.04

67.1

6.69

0.07

5.4

1330.4

4.97

GA

 

3.

0.38

6.4

0.41

33.1

3.30

0.42

22.7

745.4

2.78

Unknown

 

4.

0.47

15.1

0.49

57.3

5.71

0.52

16.9

1817.2

6.79

Unknown

 

5.

0.62

27.2

0.66

74.5

7.42

0.70

17.3

3219.2

12.02

Unknown

 

6.

0.76

28.3

0.85

75.4

7.51

0.88

3.9

8046.6

22.58

Unknown

 

 


CONCLUSIONS:

This study reveals phytochemical profiling of Neolamarckia cadamba along with quantification of quercetin and gallic acid to set its therapeutic role for further studies in various diseases.

 

ACKNOWLEDGEMENTS:

Author is thankful to NBRI, Lucknow for assistance in research work.

 

CONFLICT OF INTEREST:

Authors declare no competing interest at all.

 

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Received on 25.03.2023 Modified on 03.07.2023

Accepted on 22.10.2023 RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(1):271-276.

DOI: 10.52711/0974-360X.2024.00042