Development of Finger Print Profiles for Androgrphis echioides  Nees. and Andrographis paniculata Nees.

 

Shakila R.*, Elankani P., Jegajothipandian S.

Siddha Central Research Institute (Central Council for Research in Siddha) Arignar Anna Hospital Campus, Arumbakkam, Chennai-600 106, Tamil Nadu, India.

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

 

ABSTRACT:

Andrographis echiodes Nees and Andrographis paniculata Nees are two species of the Genera Andrographis of the Acanthaceae family. The whole plant of A. echiodes and A. paniculata find the place in the Indian system of medicine. The TLC and HPTLC finger print of different extracts were developed. The TLC and HPTLC of hexane, chloroform, ethyl acetate and ethyl alcohol extracts were carried out and compared. The TLC photo documentation at UV 254 nm, 366 nm and post derivatization with vanillin-sulphuric acid reagent revealed that the two species are composed of different type  of compounds and the HPTLC finger print also confirmed the dissimilarity between the species. These photo documentation and finger prints can be used as a reference documents for the identification as well as quality control of the two drugs in their different forms.

 

KEYWORDS: Gopuram Thangi, Nilavembu, Bhunimba, Echioidinin, Andrographolide.

 

 


1. INTRODUCTION:

A. echioides (L.) Nees is known as Gopuram Thangi in Tamil. A. paniculata Nees. is known as Nilavembu in Tamil and Bhunimba in Sanskrit. Both A. echioides and A. paniculata  are annual herbs. They are found in South India and used for curing fevers1,2. Echioidinin,3,4,5 dihydroechioidinin, echioidinin, echioidin, skullcapflavone I 2'-O-methyl ether, skullcapflavone I 2'-O-glucoside,5 androechin, echioidinin 5-O-glucoside,6 2'-oxygenated flavonoids and phenyl glycosides7 were reported from A. echioides; andrographolide, neoandrographolide,8,9 ninandrographolide,10  isoandrographolide, andropanolide11 and many other andrographolide were reported from A. paniculata. Andrographolide, the bitter principle present in A. paniculata was proven as a potent antipyretic compound. This active principle is not present in A. echioides but is used as an antipyretic. Authors aim to compare the two species by developing TLC photo documentation and high performance thin layer chromatographic finger printing.

 

2. MATERIALS AND METHODS:

2.1. Plant material 

The whole plants were collected from Mettur, Tamil Nadu in the month of May and authenticated from Botanical Survey of India, Coimbatore. The specimen samples were deposited in the Institute.

 

The samples were shade dried, powdered and stored in air tight containers for the study.

 

2.2. Chemicals

The solvents hexane, chloroform, ethyl acetate, ethyl alcohol, methanol used for the study were of AR grade. Vanillin (1 g) dissolved in ethanol sulphuric acid mixture with the ratio of  95:5, v/v was used as the visualizing agent.

 

2.2. Sample preparation

The coarsely powdered drug was weighed accurately about 4 g each and extracted successively with hexane, chloroform, ethyl acetate and ethyl alcohol using Soxhlet extractor. The extracts were concentrated by distillation and made up to 10 ml in standard flasks with respective solvents.

 

2.3. TLC plate

Aluminium plate precoated with silica gel 60F254 (Merck) of 0.2 mm thickness was  used for application and development.

 

2.4. Solvent system

Thin layer chromatographic plates with good separation of compounds were achieved after trial with different solvent systems. For the hexane extract, the solvent system of Toluene: Ethyl acetate (4.5: 0.5, v/v) was found to be suitable; similarly for chloroform extract Toluene: Ethyl acetate (5: 1, v/v), for ethyl acetate and ethyl alcohol extracts Ethyl acetate: Methanol (10 : 1, v/v) was chosen as suitable solvent system.

 

2.5. Instrument

The twin trough chamber (CAMAG) was used for developing the TLC plate. With the aid of  Linomat IV (CAMAG, Muttenz, Switzerland) applicator 10 mm bands of extracts with a distance of 10 mm were applied on 5 x10 cm plate.  CAMAG TLC scanner 030618 attached with WINCATS software were used for finger print analysis under UV 254 nm. CAMAG visualizer was used for photo documentation at UV 254 nm, 366 nm; and in visible lights after dipping in vanillin-sulphuric acid reagent followed by heating in an air circulated oven till the development of coloured spots.

 

3. RESULTS:

The thin layer chromatography (TLC) pattern of hexane extract of A. echioides (AEH) and that of A. paniculata  (APH) are shown in Fig. 1a,b,c.  The high performance thin layer chromatography (HPTLC) finger printing of AEH and APH are shown in Fig. 2 and 3; the 3D densitometric chromatogram of both extracts is shown in Fig. 4. The Rf values and area of peaks of hexane extract detected while scanning at UV 254 nm are shown in Table 1.

 

The TLC pattern of chloroform extract of A. echioides (AEC) and that of A. paniculata (APC) are shown in Fig. 5a,b,c.   The HPTLC finger printing of AEC and APC are shown in Fig. 6 and 7; the 3D densitometric chromatogram of both extracts is shown in Fig. 8. The Rf values and area of peaks of chloroform extracts detected when scanned at UV 254 nm are shown in Table 2.

 

The TLC pattern of ethyl acetate extract of A. echioides (AEEA) and that of A. paniculata (APEA) are shown in Fig. 9a,b,c. The HPTLC finger printing of AEEA and APEA are shown in Fig. 10 and 11; the 3D densitometric chromatogram of both extracts is shown in Fig. 12. The Rf values and area of peaks of ethyl acetate extracts detected when scanned at UV 254 nm are shown in Table 3.

 

The TLC pattern of ethyl alcohol extract of A. echioides (AEE) and that of A. paniculata (APE) are shown in Fig. 13a,b,c.   The HPTLC finger printing of AEE and APE are shown in Fig. 14 and 15; the 3D densitometric chromatogram of both extracts is shown in Fig. 16. The Rf values and area of peaks of chloroform extracts detected when scanned at UV 254 nm are shown in Table 4.

 

4. DISCUSSION:

TLC pattern (Fig. 1a,b,c)  of AEH at UV 254 nm showed 6 spots at Rf  0.23, 0.35, 0.45, 0.50, 0.58, 0.92 (all green) and that of APH showed only two spots at Rf 0.45 and 0.58 (both green). At  366 nm, AEH showed 6 spots at Rf 0.26, 0.35, 0.43, 0.53, 0.63 and 0.75 (all pale blue); APH showed 6 spots at 0.43 (pale blue), 0.53 (bluish brown), 0.62 (bluish brown), 0.73 and 0.85 (both bluish brown). After derivatization, AEH showed 3 spots at Rf 0.35 and  0.47 (both brown).


 

HPTLC finger print of AEH (Fig. 2) and APH (Fig. 3) showed 14 peaks. The Rf  and percentage area of peaks  are shown in Table 1. The major peaks of AEH appeared at 0.77 (32.63 %), 0.40 (16.23 %), 0.66 (12.40 %), 0.45 (11.26 %). The percentage area of  peaks at Rf  0.52 and 0.86 were 8.75 % and 6.19 % respectively. The individual contribution of  other spots ranged from 0.13 to 2.32 %. The major peaks of APH appeared at 0.56 (32.43 %) and 0.89 (13.21 %). The other peaks appearing at Rf 0.80 (8.71 %), 0.40 (6.89 %), 0.13 (6.62 %), 0.50 (6.62 %), 0.14 (5.98 %) were individually contributing >5% to the total area of the separated peaks.  The percentage area of all other peaks <5 % only.  Though both the extracts showed 14 peaks, the major peaks of AEH and APH are entirely different and Rf of other peaks are also different which is evident from the 3D chromatogram (Fig. 4).

 

Figure 2. HPTLC densitometric chromatogram of hexane extract of A. echiodes

 

Figure 3. HPTLC densitometric chromatogram of hexane extract of A. paniculata

 

Figure 4. HPTLC densitometric 3D chromatogram of hexane extract of A. echiodes and A. paniculata

 

Table 1. Rf value and percentage area of peaks of hexane extracts at UV 254 nm

 

Sl.No

AEH

APH

 

Rf

% Area of peak

Rf

% Area of peak

 

1.               

0.06

0.13

0.13

6.62

 

2.               

0.10

0.14

0.14

5.98

 

3.               

0.19

2.32

0.22

1.92

 

4.               

0.29

2.29

0.31

4.05

 

5.               

0.32

2.09

0.40

6.89

 

6.               

0.40

16.23

0.50

6.62

 

7.               

0.45

11.26

0.56

32.43

 

8.               

0.52

8.75

0.66

3.36

 

9.               

0.62

4.13

0.72

3.55

 

10.             

0.66

12.40

0.80

8.71

 

11.             

0.77

32.63

0.83

4.56

 

12.             

0.86

6.19

0.89

13.21

 

13.             

0.94

0.86

0.92

1.65

 

14.             

0.97

0.57

0.97

0.65

 

TLC (Fig. 5) of AEC  at UV 254 nm showed 5 spots at Rf 0.43, 0.52, 0.64, 0.71 and 0.79 (all green); APC showed two spots at Rf 0.07 and 0.79 (both green). At 366 nm, AEC showed 8 spots at Rf 0.07 (pale pink), 0.19 (pink), 0.33 (pink), 0.43 (brown),  0.57 (pink), 0.66 (pale blue), 0.72 (pink), 0.79 (brown); APC showed 7 spots with Rf 0.07 (pink), 0.17 (pink), 0.31 (pink), 0.39 (pink), 0.66 (blue), 0.72 (pink) and 0.79 (brown). The spot at Rf 0.43  of AEC is not seen in APC.   After derivatization, AEC showed 6 spots at Rf 0.07 (purple), 0.16 (grey), 0.48 (violet), 0.64 (yellowish brown), 0.72 (purple) and 0.81 (violet); APC showed 4 spots at Rf 0.07 (violet), 0.48 (violet). 0.64 (violet) and 0.79 (violet). The spots at Rf 0.64 of both APH and APC are not same compounds.

 



 

 


The HPTLC of AEC (Fig. 6) showed 15 peaks in which the peak at Rf 0.40 (33.61 %), 0.58 (20.77 %), 0.73 (14.39 %), 0.65 (14.15) and 0.47 (6.08 %) were the major. All other peaks were minor. The HPTLC of APC (Fig. 7) showed 21 peaks out of which only 3 peaks at Rf 0.06 (30.79 %), 0.73 (21.48%) and 0.94 (16.76%) were major. The peak at Rf 0.58 contributed 5.29 % to the total area. All other peaks were minor with less than 5 % of total area. Both extracts showed peak at Rf 0.7 with different peak area. The Rf  and percentage area of all the peaks  are shown in Table 2. The dissimilarity of both extracts were seen through the 3D chromatogram (Fig. 8).

 

 

Figure 6. HPTLC densitometric chromatogram of chloroform extract of A. echiodes

 

 

Figure 7. HPTLC densitometric chromatogram of chloroform extract of A. paniculata

 

 

Figure 8. HPTLC densitometric 3D chromatogram of chloroform extract of A. echiodes and A. paniculata


 

Table 2. Rf value and percentage area of peaks of chloroform extracts at UV 254 nm

Sl.No

AEC

APC

Rf

% Area of peak

Rf

% Area of peak

1.               

0.06

0.47

0.06

30.79

2.               

0.09

0.27

0.10

0.40

3.               

0.13

0.42

0.14

1.17

4.               

0.17

0.67

0.16

0.25

5.               

0.25

1.50

0.18

0.24

6.               

0.40

33.61

0.20

0.72

7.               

0.47

6.08

0.30

3.37

8.               

0.58

20.77

0.35

0.73

9.               

0.65

14.15

0.37

1.17

10.             

0.73

14.39

0.40

0.83

11.             

0.83

1.54

0.41

1.04

12.             

0.84

1.37

0.44

1.56

13.             

0.88

0.22

0.45

1.41

14.             

0.90

0.29

0.48

2.03

15.             

0.95

4.25

0.53

2.61

16.             

 

 

0.58

5.29

17.             

 

 

0.66

3.92

18.             

 

 

0.73

21.48

19.             

 

 

0.86

0.57

20.             

 

 

0.94

16.76

21.             

 

 

0.99

3.56

 

TLC (Fig. 9) of AEEA  at UV 254 nm showed 5 spots at Rf 0.22, 0.38, 0.57, 0.69 and 0.83 (all green); APEA showed a spot at Rf 0.83 (green); in addition to this spots, other spots of AEEA are also seen in APEA in very low concentration. At 366 nm, AEEA showed 6 spots at Rf 0.26 (creamy blue), 0.33 (pale blue), 0.38 (pale blue), 0.53 (brown), 0.60 (pale blue) and 0.79 (brown)); APEA showed 5 spots with Rf 0.24 (creamy blue), 0.33 (pale blue), 0.52 (pale blue), 0.67 (pale blue) and 0.87 (blue). The spot at Rf 0.79 of AEC and APC are not one and same compound. After derivatization, AEEA showed 5 spots at Rf 0.10 (brown), 0.24 (brown), 0.34 (brown), 0.53 (brown) and 0.66 (brown). APEA showed 3 spots at Rf 0.10 (grey), 0.21 (grey) and 0.79 (violet).

 

 

The HPTLC of AEEA (Fig. 10) showed 7 peaks at Rf values 0.54 (25.85 %), 0.90 (24.68 %), 0.23 (16.64 %), 0.76 (10.87 %), 0.85 (8.72 %), 0.33 (8.23 %) and 0.64 (5.01 %).  The HPTLC of APEA (Fig. 11) showed 11 peaks at Rf values 0.78 (25.69 %), 0.93 (23.07 %), 0.89 (18.23 %) and 0.84 (11.06 %).  The peak at Rf value 0.34 contributed to 5.39 % and other peaks were minor. The Rf values and the area of all peaks are shown in Table 3. The 3D chromatogram (Fig. 12) also showed that the HPTLC pattern of both extracts was differing from each other

 

Table 3. Rf value and percentage area of peaks of ethyl acetate extracts at UV 254 nm

Sl.No

AEEA

APEA

Rf

% Area of peak

Rf

% Area of peak

1.        

0.23

16.64

0.13

3.17

2.               

0.33

8.23

0.18

2.49

3.               

0.54

25.85

0.24

4.75

4.               

0.64

5.01

0.34

5.39

5.               

0.76

10.87

0.47

2.58

6.               

0.85

8.72

0.49

3.19

7.               

0.90

24.68

0.57

0.37

8.               

 

 

0.78

25.69

9.               

 

 

0.84

11.06

10.             

 

 

0.89

18.23

11.             

 

 

0.93

23.07

 

TLC (Fig. 13) of AEE at UV 254 nm showed 4 spots at Rf 0.26, 0.38, 0.52 and 0.86 (all green); APE showed a spot at Rf 0.78 (green). At 366 nm, AEE showed 2 spots at Rf 0.26 (creamy blue) and 0.38 (pale blue); APE showed a spot at Rf 0.34 (creamy blue). After derivatization, AEE  showed 3 spots at Rf 0.09 (brown), 0.26 (brown) and 0.53 (brown). APE also showed 3 spots at Rf 0.09 (grey), 0.24 (grey) and 0.78 (grey).

 

Figure 10. HPTLC densitometric chromatogram of ethyl acetate extract of A. echiodes

 

Figure 11. HPTLC densitometric chromatogram of ethyl acetate extract of A. paniculata



Figure 12. HPTLC densitometric 3D chromatogram of ethyl acetate extract of  A. echiodes and A. paniculata


 

The HPTLC finger print of AEE (Fig. 14) showed 12 peaks in which the peak at Rf  0.73 was the major of all the peaks. The other major peaks were 0.31 (16.23 %), 0.61 (12.40 %), 0.37 (11.26 %), 0.45 (8.75 %), 0.85 (6.19 %) and  0.56 (4.13 %). Other peaks were minor.  The HPTLC finger print of AEP (Fig. 15) showed 13 peaks of which the peak at Rf

 

 

0.50 (32.43 %) and 0.87 (13.21 %) were the major peaks. The peaks at Rf value 0.77 (8.71 %), 0.32 (6.89 %), 0.42 (6.62 %) and 0.02 (5.98 %) were individually contributing more than 5 % to the total peak area and other peaks were minors. The Rf value and the peak area are shown in Table 4. The 3D densitometric chromatogram (Fig. 16) showed the difference between the two extracts.


 

 


Figure 14. HPTLC densitometric chromatogram of ethanol extract of A. echioides

 

Figure 15. HPTLC densitometric chromatogram of ethyl acetate extract of A. paniculata

 

Figure 16. HPTLC densitometric 3D chromatogram of ethanol extract of  A. echiodes and A. paniculata

Table 4. Rf value and percentage area of peaks of ethyl alcohol extracts at UV 254 nm

Sl.No

AEE

APE

Rf

% Area of peak

Rf

% Area of peak

1.          1

0.08

2.32

0.02

5.98

2.               

0.19

2.29

0.11

1.92

3.               

0.22

2.09

0.22

4.05

4.               

0.31

16.23

0.32

6.89

5.               

0.37

11.26

0.42

6.62

6.               

0.45

8.75

0.50

32.43

7.               

0.56

4.13

0.61

3.36

8.               

0.61

12.40

0.68

3.55

9.               

0.73

32.63

0.77

8.71

10.             

0.85

6.19

0.81

4.56

11.             

0.93

0.86

0.87

13.21

12.             

0.97

0.57

0.91

1.65

13.             

 

 

0.97

0.46

 

CONCLUSION:

The generated TLC photo documentation patterns and the HPTLC finger print profiles of different extracts will be helpful for authentication of A. echioides as well as A. paniculata in any formulation and in powder form.

 

REFERENCES:

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10.     Meng Z. Isolation and identification of a new glucoside-bicyclic diterpenoid lactone from Andrographis paniculata Nees.  Nanjing Yaoxueyuan Xuebao. (1): 1982; 15-20. Chem Abstr. 99: 1983; 191697j.

11.     Pramanick S, Banerjee S, Achari B, Das B, Sen AK Sr, Mukhopadhyay S, Neuman A,  Prange T.  Andropanolide and isoandrographolide, minor diterpenoids from Andrographis  paniculata: structure and X-ray crystallographic analysis.    J Nat Prod. 69: 2006; 403-405.  

 

 

Received on 13.10.2013       Modified on 30.11.2013

Accepted on 15.12.2013      ©A&V Publications All right reserved

Res.  J. Pharmacognosy & Phytochem. 6(1): Jan.-Mar. 2014; Page 22-29