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:
1. Kirtikar, K.R.; Basu, B.D.
Indian Medicinal Plants; Periodical Experts Book Agency, New Delhi,
India. 1975; Volume 3: 1884-1886.
2. Chopra, R.N.; Nayer,
S.L.; Chopra, I.C. Glossary of Indian Medicinal Plants; Council of
Scientific and Industrial Research: New Delhi, India. 1980; 18.
3. Govindachari TR, Parthasarathy PC, Pai BR, Subramaniam PS. Chemical
examination of Andrographis echioides-I:
structure and synthesis of echioidinin.
Tetrahedron. 21(9); 1965: 2633-2640.
4. Govindachari TR, Parthasarathy PC, Pai BR, Subramaniam PS. Chemical
examination of Andrographis echioides-II:
structure and synthesis of echioidin. Tetrahedron. 21(12); 1965: 3715-3720.
5. Jayaprakasam B, Damu AG, Gunasekar D, Blond A,
Bodo B. Dihydroechioidinin,
a flavanone from Andrographis echioides. Phytochemistry. 52(5);
1999: 935-937.
6. Jayaprakasam B, Gunasekar D, Rao KV, Blond A,
Bodo B. Androechin, a new chalcone glucoside from Andrographis echioides. J
Asian Nat Prod Res. 3(1); 2001: 43-48.
7. Shen DY, Juang SH, Kuo PC, Huang GJ,
Chan YY, Damu AG, Wu TS. Chemical constituents
from Andrographis
echioides
and their anti-inflammatory activity. Intl J Mol Sci. 14(1); 2013: 496-514.
8. Fujito T, Fujitani R,
Takeda Y, Takaishi Y, Yamada T, Kido M, Miura I. on
the diterpenoids of Andrographis paniculata: X-ray crystallographic
analysis of andrographolide and structure
determination of new minor diterpenoids. Chem Pharm Bull. 32; 1984: 2117.
9. Chen LX, Qu GX, Qiu F. Studies on diterpenoids
from Andrographis paniculata. Zhongguo Zhong Yao Za Zhi. 31; 2006: 1594-1597.
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
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