This document is the presentation slide for viva. Its the summary of my research.

1. ANTIOXIDANT PROPERTIES IN CALLUS
CULTURES AND IN INTACT PLANT PARTS
OF
GYNURA PROCUMBENS
VIJENDREN A/L KRISHNAN
GS21956
SUPERVISORY COMMITTEE:
PROF. DR. MAZIAH MAHMOOD
DR. SYAHIDA AHMAD
1

2.  Gynura procumbens have been used in folk medicine
since ages, however there is no report on quantitative
analysis of antioxidant properties in all intact plant parts
and also in in-vitro cultures of G. procumbens.
 Tremendous development in herbal industry, requires
more valuable extracts
 Hence, this research will provide method to produce
high amount of cell cultures and lead to improvisation
of the antioxidant properties in the cultures.
2
Problem Statement & Significance

3. OBJECTIVE
 To compare the antioxidant properties in intact plant
parts of Gynura procumbens and Gynura bicolor.
 To study antioxidant properties of Gynura
procumbens’ leaf, stem and root derived callus.
 To establish callus induction and callus proliferation
from leaf, stem and root explants of Gynura
procumbens.
3

4. 4
 In Thailand, used to treat topical inflammation, rheumatism,
and viral ailments on skin.
 In Indonesia, used to treat fever, rashes, remedy for
ringworm infection. (Iskander et al., 2002)
 Gynura procumbens posses anti-inflammatory, anti-
hyperlipidaemic, anti-hypertensive, anti-cancer, and anti-
ulcerogenic properties (Zhang and Tan, 2000; Iskander et al.,
2002; Kim et al., 2006, Jenie and Meiyanto, 2008)
 Presence of essential oils, steroids, bitter principles,
flavonoids, valepotriates, coumarins, and alkaloid were
reported in various organic solvents by TLC. (Iskander et
al., 2002)
Review literature

5.  Kingdom : Plantae
 Division : Magnoliophyta
 Class : Magnoliopsida
 Order : Asterales
 Family : Asteraceae (alt. compositae)
 Tribe : Senecioneae
 Genus : Gynura
 Species : procumbens
5
(Taxonomy – Botanica Sistematica Online, 2009;
USDA,ARS, National Genetic Resources
Program 2011)

6. 6
Component Content
Moisture 7.08%
Carbohydrate 0.1968 µg glu/ 100g DW
Protein 4.51 g/ 100 g DW
Lipid 0.023 g/ 100 g DW
Abdel-Wahab et al., 2009;Puangpronpitag et al., 2010
Elements Content (%)
C 44.36
O 39.92
Mg 0.4
P 0.4
S 0.39
Cl 3.63
K 8.7
Al 0.06
Ca 1.73

7. 7

8. 8
DPPH (BrandWilliamsetal.,1995)
Figure 1: Total antioxidant content of aerial parts of G. procumbens (P)
and G. bicolor (B) expressed as percent of inhibition using DPPH
method. The bar indicates the standard deviation of mean (n = 3)]

9. 9
FRAP (BenzieandStrain, 1999)
Figure 2: Total antioxidant content of aerial parts of G. procumbens (P)
and G. bicolour (B) expressed as percent of inhibition using FRAP
method. The bar indicates the standard deviation of mean (n = 3)

10. 10
Total Phenolic Content (Singleton and Rossi, 1965)
Figure 3: Total phenolic content of aerial parts of G. procumbens (G) and
G. bicolor (B) expressed as gallic acid equivalent (mg/g FW). The bar
indicates the standard deviation of mean (n = 3).

11. 11
Total Flavonoid Content (Zhishenetal.,1999)
Figure 4: Total flavonoid content in aerial parts of G. procumbens (G)
and G. bicolor (B) expressed as kaempferol equivalent (µg/g FW). The
bar indicates the standard deviation of mean (n = 3)

12. 12
Figure 5: Total ascorbic acid content in aerial parts of G. procumbens (G)
and G. bicolor (B) expressed as ascorbic acid equivalent (µg/g FW). The
bar indicates the standard deviation of mean (n = 3)
Ascorbic acid content (DaviesandMasten,1990)

13. 13
Thin Layer Chromatography
Figure 6: Thin layer chromatography performed using ethyl acetate/formic acid/
acetic acid/water, 100:11:11:26 (V/V) as solvent. [(C) Catechin, (H) Hesperitin,
(K) Kaempferol, (N) Naringenin, (Q) Quercetin, (R) Rutin, (PR) G. procumbens root,
(PS) G. procumbens stem, (PL) G. procumbens leaf, (BR) G. bicolor root, (BS)
G. bicolor stem, (BL) G. bicolor leaf]

14. Callus induction of G. procumbens
leaf, stem and root
Callus proliferation of G. procumbens
leaf, stem and root
Callus proliferation profile of
G. procumbens leaf, stem and root
14
TISSUE CULTURE

15. Figure7: Influenceof auxinson number of daysto callus initiationon G.
procumbens leaf, stem and root explants.The results indicate mean
standarddeviation(SD). N=10.
15

16. 16
Figure8: Callusinitiationfrom leaf on MS medium supplementedwith 5µM of
variousauxinsafter 30 daysof culture: (A) 2,4-D, (B) Dic, (C) IAA, (D) IBA, (E)
NAA, (F) Pic, (G) MSO. (Bar indicates1 cm)
A B C
D E F
G

17. A B C
D E F
G
17
Figure9: Callusinitiationfrom stem on MS medium supplementedwith 5µM
of variousauxinsafter 30 daysof culture: (A) 2,4-D, (B) Dic, (C) IAA, (D)
IBA, (E) NAA, (F) Pic, (G) MSO. (Bar indicates1 cm).
E

18. 18
Figure10 : Callusinitiationfrom root on MS medium supplementedwith 5µM of
variousauxinsafter 30 daysof culture: (A) 2,4-D, (B) Dic, (C) IAA, (D) IBA, (E)
NAA, (F) Pic, (G) MSO. (Bar indicates1 cm).
A B C
D E F
G

19. 19
Figure11: : Influence of variousauxinson callus initiationof G. procumbens
leaf after three sub-culturesfor 30 dayseach passage.Meanswithin columns
followedby the same alphabetsare not significantlydifferent at p<0.05
(Duncan’stest)

20. 20
Figure12: : Influence of variousauxinson callus initiationof G.
procumbensstem after three sub-culturesfor 30 dayseach passage.Means
withincolumns followed by the same alphabetsare not significantly
differentat p<0.05(Duncan’stest)

21. 21
Figure13: : Influence of variousauxinson callus initiationof G.
procumbensroot after three sub-culturesfor 30 dayseach passage.Means
withincolumns followed by the same alphabetsare not significantly
differentat p<0.05(Duncan’stest)

22. Callus proliferation
22

23. 23
Figure 14: Gynura procumbens’ leaf callus proliferation on
various auxins after 30 days of culture. Means within columns
followed by the same alphabets are not significantly different at
p<0.05 (Duncan’s test).

24. 24
Figure 15: Gynura procumbens’ stem callus proliferation on
various auxins after 30 days of culture. Means within columns
followed by the same alphabets are not significantly different at
p<0.05 (Duncan’s test).

25. 25
Figure 16: Gynura procumbens’ root callus proliferation on
various auxins after 30 days of culture. Means within columns
followed by the same alphabets are not significantly different at
p<0.05 (Duncan’s test).

26. 26
Figure 17: Leaf derived callus proliferated with auxin treatments
after 30 days of culture: (A) 2,4-D, (B) Dic, (C) NAA, (D) PIC.
(Bar indicates 1 cm).
A B
C D

27. 27
Figure 18: Stem derived callus proliferated with auxin treatments
after 30 days of culture. (A) 2,4-D, (B)Dic, (C) IAA, (D) IBA, (E)
NAA, (F) Pic. [Bar indicates 1 cm]
A B C
D E F

28. 28
A B
C D
Figure 19: Root derived callus proliferated with auxin treatments
after 30 days of culture. (A) 2,4-D, (B) Dic, (C) NAA, (D) Pic.
[Bar indicates 1 cm].

29. 29
Figure 20: Influence of 2,4-D concentrations on callus
multiplication of G. procumbens leaf. Means within columns
followed by the same alphabets are not significantly different at
p<0.05 (Duncan’s test)

30. 30
Figure 21: Effect of 2,4-D concentrations on callus multiplication
of G. procumbens stem. Means within columns followed by the
same alphabets are not significantly different at p<0.05 (Duncan’s
test).

31. 31
Figure 22: Effect of 2,4-D concentrations on callus multiplication
of G. procumbens root. Means within columns followed by the
same alphabets are not significantly different at p<0.05 (Duncan’s
test).

32. 32
Table 1: Callus proliferation from leaf explants of G. procumbens
on MS media supplemented with 10 µM 2,4-D with various
concentrations of cytokinins after 30 days of culture.

33. 33
Table 2: Callus proliferation from stem explants of G. procumbens
on MS media supplemented with 15 µM 2,4-D with various
concentrations of cytokinins after 30 days of culture

34. 34
Table 3: Callus proliferation from root explants of G. procumbens
on MS media supplemented with 15 µM 2,4-D with various
concentrations of cytokinins after 30 days of culture.

35. 35
Figure 23 : Influence of 2,4-D (10 µM) in combination with BAP
(7.5 µM) on callus multiplication of G. procumbens leaf. Means
within columns followed by the same alphabets are not
significantly different at p<0.05 (Duncan’s test).

36. 36
Figure 24 : : Influence of 2,4-D (15 µM) in combination with
BAP (7.5 µM) on callus multiplication of G. procumbens stem.
Means within columns followed by the same alphabets are not
significantly different at p<0.05 (Duncan’s test).

37. 37
Figure 25 : : Effect of 2,4-D (10 µM) in combination with BAP
(5.0 µM) on callus multiplication of G. procumbens root. Means
within columns followed by the same alphabets are not
significantly different at p<0.05 (Duncan’s test).

38. 38
Conclusions
 Gynura procumbens root extract exhibit highest
antioxidant activity in all assays.
 TLC analysis reveal presence of kaempherol in G.
procumbens leaf extract and rutin in G. procumbens
root extract.
 MS medium augmented with 5 µM 2,4-D was optimum
for callus initiation from G. procumbens leaf and root
explant.
 MS medium augmented with 5 µM Pic was optimum
for callus initiation from G. procumbens stem explant.

39. 39
 Leaf derived callus proliferated optimally on MS
medium supplemented with 10 µM 2,4-D and 7.5 µM
BAP.
 Stem derived callus proliferated optimally on MS
medium supplemented with 15 µM 2,4-D and 7.5 µM
BAP.
 Root derived callus proliferated optimally on MS
medium supplemented with 10 µM 2,4-D and 5.0 µM
BAP.
 Leaf derived callus of 21 days old was suitable for sub-
culturing.
 Stem and root derived callus of 24 days old were
optimum for sub-culturing.

40. 40
Publications and Conferences
 Vijendren K., Syahida A., and Maziah M. Callus
Induction of Selected Malaysian Medicinal Plants.
Malaysian Society of Applied Biology Conference, 13-
15 June 2010, Kota Bharu, Kelantan, Malaysia
 Krishnan Vijendren, Ahmad Syahida and Mahmood
Maziah, Evaluation of antioxidant potential in intact
plant parts and callus of Gynura procumbens and intact
parts of Gynura bicolor. (DRAFT)

41. 41
References
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(2009). Energy-dispersive X-ray microanalysis of elements’ content of medicinal
plants used traditionally as anticancer cure. Research Journal of Biological Sciences,
4 (5): 547-549.
 Benzie, I. F., and Strain, J. J. (1996). Ferric reducing ability of plasma (FRAP) as a
measure of “antioxidant power”: the FRAP assay. Analytical Biochemistry, 239: 70-
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 Brand-Williams, W., Cuvelier, M. E., and Berset, C. (1995). Use of free radical
method to evaluate antioxidant activity. Lebensmittel Wissenschaft und Technologie,
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 Davies, S. H. R., and Masten, S. J. (1991). Spectrophotometric method for ascorbic
acid using dichlorophenolindophenol: elimination of the interference due to iron.
Analytica Chimica Acta, 248: 225-221.
 Iskander, M. N., Song, Y., Coupar, I. M., and Jiratchariyakul, W. (2002). Anti-
inflammatory screening of the medicinal plant Gynura procumbens. Plant foods for
human nutrition, 57: 233-244.
 Jenie, R. I., and Meiyanto, E. (2008). Combination of sambung nyawa (Gynura
procumbens (Lour.) Merr.) leaves ethyl acetate fraction (SEF)-doxorubicin (dox)
induces apoptosis in human breast cancer T47D cells. Proceeding Molecular
Targeted Therapy Symposium 2008.

42. 42
 Kim, M. J., Lee, H. J., Wiryowidago, S., and Kim, H. K. (2006). Anti-hypertensive
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of Medicinal Food, 9 (4): 587-590.
 Luigi, R. (2009). Botanica Sistematica. Taxonomy – Botanica Sistematica Online
–(2009). Retrieved: March 23 2012, from,
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 Singleton, V. L., and Rossi, J. A. Jr. (1965). Colorimetry of total phenolics with
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 USDA, ARS, National Genetic Resources Program. Germplasm Resources
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http://www.ars-grin.gov/cgi-bin/npgs/html/taxon.pl?104260
 Zhang, X. F., and Tan, B. K. H. (2000). Effects of an ethanolic extract of Gynura
procumbens on serum glucose, cholesterol and triglyceride levels in normal and
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43. THANKS
43

44. 44
2,4-D
DIC
IAA
IBA NAA PIC
Callus initiation from leaf (10days)

45. 45
2,4-D DIC
IAA
IBA NAA PIC
Callus initiation from leaf (30days)

46. 46
2,4-D
DIC
IAA
IBA NAA PIC
Callus initiation from stem (10 days)

47. 47
2,4-D
DIC
IAA
IBA NAA PIC
Callus initiation from stem (30days)

48. 48
2,4-D DIC IAA
IBA NAA PIC
Callus initiation from root (20days)

49. 49
2,4-D
DIC
IAA
IBA NAA PIC
Callus initiation from root (30 days)

50. 50
Figure : Effect of various concentration of cytokinin with 10µM 2,4-D
for G. procumbens leaf callus proliferation at 30 days of culture

51. 51
Figure : Effect of various concentration of cytokinin with 10µM 2,4-D
for G. procumbens stem callus proliferation at 30 days of culture

52. 52
Figure : Effect of various concentration of cytokinin with 10µM 2,4-D
for G. procumbens root callus proliferation at 30 days of culture