ANTIOXIDANT PROPERTIES IN CALLUS CULTURES AND IN INTACT PLANT PARTS OF GYNURA PROCUMBENS

VIJENDREN A/L KRISHNANGS21956

SUPERVISORY COMMITTEE:

PROF. DR. MAZIAH MAHMOODDR. SYAHIDA AHMAD

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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.

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Problem Statement & Significance

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.

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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

Kingdom : Plantae Division : Magnoliophyta Class : Magnoliopsida Order : Asterales Family : Asteraceae (alt. compositae) Tribe : Senecioneae Genus : Gynura Species : procumbens

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(Taxonomy – Botanica Sistematica Online, 2009;USDA,ARS, National Genetic Resources Program 2011)

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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

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Antioxidant

DPPH

FRAP

TPCTFC

Ascorbic acid

content

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DPPH (Brand

Williams et al., 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)]

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FRAP (Benzie and Strain , 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)

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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).

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Total Flavonoid Content (Zhishen et al., 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)

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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 (Davies and Masten, 1990)

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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] 

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

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TISSUE CULTURE

Figure 7: Influence of auxins on number of days to callus initiation on G. procumbens leaf, stem and root explants. The results indicate mean ± standard deviation (SD). N=10.

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Figure 8: Callus initiation from leaf on MS medium supplemented with 5µM of various auxins after 30 days of culture: (A) 2,4-D, (B) Dic, (C) IAA, (D) IBA, (E) NAA, (F) Pic, (G) MSO. (Bar indicates 1 cm)

A B C

D E F

G

A B C

D E F

G

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Figure 9: Callus initiation from stem on MS medium supplemented with 5µM of various auxins after 30 days of culture: (A) 2,4-D, (B) Dic, (C) IAA, (D) IBA, (E) NAA, (F) Pic, (G) MSO. (Bar indicates 1 cm).

E

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Figure 10 : Callus initiation from root on MS medium supplemented with 5µM of various auxins after 30 days of culture: (A) 2,4-D, (B) Dic, (C) IAA, (D) IBA, (E) NAA, (F) Pic, (G) MSO. (Bar indicates 1 cm).

A B C

D E F

G

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Figure 11: : Influence of various auxins on callus initiation of G. procumbens leaf after three sub-cultures for 30 days each passage. Means within columns followed by the same alphabets are not significantly different at p<0.05 (Duncan’s test)

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Figure 12: : Influence of various auxins on callus initiation of G. procumbens stem after three sub-cultures for 30 days each passage. Means within columns followed by the same alphabets are not significantly different at p<0.05 (Duncan’s test)

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Figure 13: : Influence of various auxins on callus initiation of G. procumbens root after three sub-cultures for 30 days each passage. Means within columns followed by the same alphabets are not significantly different at p<0.05 (Duncan’s test)

Callus proliferation

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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).

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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).

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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).

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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

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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

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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].

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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)

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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).

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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).

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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.

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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

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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.

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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). 

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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).

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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).

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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.

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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.

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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)

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References Abdel-Wahab, S. I., Ain, N. M., Abdul, A. B., Elhassan, M. M., and Ibrahim, T. A.

T. (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: 70-76.

Brand-Williams, W., Cuvelier, M. E., and Berset, C. (1995). Use of free radical method to evaluate antioxidant activity. Lebensmittel Wissenschaft und Technologie, 28: 25-30.

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.

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Kim, M. J., Lee, H. J., Wiryowidago, S., and Kim, H. K. (2006). Anti-hypertensive effects of Gynura procumbens extract in spontaneously hypertensive rats. Journal of Medicinal Food, 9 (4): 587-590.

Luigi, R. (2009). Botanica Sistematica. Taxonomy – Botanica Sistematica Online –(2009). Retrieved: March 23 2012, from, http://angr.altervista.org/botanica/hypertext/0791.htm

Puangpronpitag, D., Chaichanadee, S., Naowaratwattana, W., Sittiwet, C., Thammasarn, K., Luerang, A., and Kaewseejan, N. (2010). Evaluation of nutritional value and antioxidative properties of the medicinal plant Gynura procumbens extract. Asian Journal of Plant Science, 9 (3): 146-151.

Singleton, V. L., and Rossi, J. A. Jr. (1965). Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents. American Journal of Enology and Viticulture, 16: 144–158.

USDA, ARS, National Genetic Resources Program. Germplasm Resources Information Network - (GRIN) [Online Database] (2011). National Germplasm Resources Laboratory, Beltsville, Maryland. Retrieved: March 6, 2011, from: 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 streptozotocin-induced diabetic rats. Singapore Medical Journal, 41 (1): 9-13.

Zhishen, J., Mengcheng, T., and Jianming, W. (1999). The determination of flavonoid content in mulberry and their scavenging effects on superoxide radicals. Food Chemistry, 64: 555–559.

THANKS

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2,4-DDIC

IAA

IBA NAA PIC

Callus initiation from leaf (10days)

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2,4-D DICIAA

IBA NAA PIC

Callus initiation from leaf (30days)

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2,4-DDIC

IAA

IBA NAA PIC

Callus initiation from stem (10 days)

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2,4-DDIC

IAA

IBA NAA PIC

Callus initiation from stem (30days)

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2,4-D DIC IAA

IBA NAA PIC

Callus initiation from root (20days)

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2,4-DDIC

IAA

IBA NAA PIC

Callus initiation from root (30 days)

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

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

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