SIMON ALOO SEMINAR 2023. PPT.pdf

Simon ALOO
3rd Semester PhD course
Insights into elicitor’s role in augmenting secondary metabolites
production and improvement of health functions of plant products
1
Seminar
2023-10-05

Introduction
 Secondary metabolites and benefits
• They are chemical compounds in organisms that are
not directly involved in the normal growth and
development of the organism.
• They include alkaloids, flavonoids, allied phenolic
terpenoids, and sulphur containg compounds
including gluosinolates among others.
• They offer major component of plant defense
mechanism against pest and pathogens
• They are found only in specific organisms or
groups of organisms, and are an expression of
individuality
• In human, these compounds are known for their
health benefits such as potent antioxidant,
antiinflammation, antiobesity, antidiabetic among
others.
• Background • Material and methods • Conclusion
• Results & discussion

 Biosynthetic pathways of secondary metabolites
• These compounds are biosynthesized through series of
enzymatic reactions using simple building blocks in different
ways.
• There are several biosynthetic pathways of these compounds in
plants.
1. Shikimic acid pathway (phenylpropanoids)
2. Mavalonic acid pathway (quinones)
3. Amino acid pathway (alkaloids)
4. Acetate-malonate pathway (fatty acids, phenols, and
quinones)
5. Combined pathways.
Figure. Schematic representation of biosynthetic pathway of polyphenols. Green,
pink, yellow, purple, and blue areas depicts phenolic acids, lignans, coumarins,
stilbenes, and flavonoids, respectively

Plant tissue culture (micropropagation)
Growth of tissues or cells in an artificial
medium separate from the parent organism.
Approaches to improve secondary metabolites
Genetic engineering
Genetic modification or genetic manipulation,
is the modification and manipulation of an
organism's genes using technology.
Elicitation
Applying substances which induce
physiological changes in the plant.

Elicitation as a new perspective into plant phytonutrient

Different types of plant elicitors

Study materials

Methodology

Ultrasound treatment (200 W and for
5, 10, and 20 min at 25 ℃)
Determination of total flavonoid polyphenol contents
Determination of chlorophyll and vitamins
Determination of flavonoids
Determination of triterpenes
Tyrosine ammonia-lyase (TAL) and phenylalanine
ammonia-lyase (PAL) assays
• Background • 1ST paper Material and methods • Conclusion

DPPH and ABTS assays
C2C12 cell line
• Background • 1ST paper Material and methods • Conclusion

• Background • 1ST paper material and method: brief • Conclusion

80% ethanol, wash and rinse
Germination under hydroponic
conditions (2.0 mmol/L Ca(NO3)2, 4.0
mmol/L KNO3, 0.74 mmol/L KH2PO4, and 1.0
mmol/L MgSO4; light intensity of 300 μmol m/s;
relative humidity of 60%)
Elicitation
Transfer uniform seedlings
• Background • 2nd paper Material and methods • Conclusion
15 days, 3 days,

Ascorbic acid and reduced glutathione analysis
Gene expression
Total
polyphenols
Antioxidant
analysis
• Background • 2nd Paper Material and methods • Conclusion

• Background • 2nd paper material and method: brief • Conclusion

Table 1. Triterpene contents in Centella asiatica.
• 1st paper Results & discussion
Elicitation using ultra sound improved triterpene content of the Centella asiatica with respect to the control. The amounts of
madecassoside, asiaticoside, and madecasic acid were increased in a time-dependent manner
Glycosides Aglycones

Table 2. Functional compounds in Centella asiatica.
Total polyphenols, total flavonoids, individual flavonoids, vitamins and chlorophyll contents were enhanced after ultra sound treatment. The best time duration for ultrasound treatment was
determined to be 20 min, which induced the highest increase of total polyphenol and total flavonoid contents. Chlorophyll b content was significantly increased by ultrasound treatment for 10
min.

Fig. 1. Effect of ultrasound-treated Centella asiatica on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS)
radical scavenging activity, and reducing power. The DPPH and ABTS radical scavenging activity and reducing power are expressed as Trolox equivalent mg/100 g dry weight. Different
letters (a,b for DPPH, a′,b′,c′,d’ for ABTS, and a′′,b′′,c′′ for reducing power) above the bars indicate significant differences according to Duncan's test (p < 0.05). NB; Antioxidant potential
was significantly improved after treatment with different doses of ultra sound. Compared with that in the untreated leaves, treatment with ultrasound for 10 min significantly
increased the DPPH radical scavenging activity, ABTS radical scavenging activity, and reducing power 1.75-, 1.35-, and 1.20-fold, respectively

Fig. 2. Effect of ultrasound treated Centella asiatica on (A) catalase (CAT), (B) peroxidase (POD), and (C) phenylalanine ammonia-lyase (PAL), and (D) tyrosine ammonia-lyase (TAL)
activities. Data are presented as the mean ± standard error (n = 3). Different letters above the bars indicate significant differences at p < 0.05. The highest CAT and POD activity was observed at
10 min. Similarly, compared with that in the untreated leaves, PAL and TAL activities in C. asiatica leaves were enhanced 1.30- and 1.55-fold, respectively, after ultrasound treatment for 10 min
Stress markers
Phenolic biosynthesis markers

Fig. 3. Effect of ultrasound treated Centella asiatica extracts (50 μg/mL) on (A) cytotoxicity, (B) protective activity, (C and D) reactive oxygen species production against H2O2-induced
C2C12 cells, (E) glutathione depletion and (F) lipid peroxidation against H2O2-induced C2C12 cells. Data are presented as the mean ± standard error (n = 3). ##p < 0.01 and ###p < 0.001,
versus the control cells; *p < 0.05, **p < 0.01, and ***p < 0.001 versus the H2O2-treated cells. Con, Control; GSH, glutathione; MDA, malondialdehyde. The extracts of the ultra sound-
treated leaves did not reveal any toxicity. Indeed, they protected the cells against damage by excessive accumulation of ROS and enhancing the activity of antioxidant-related
enzymes
• 1st Paper Results & discussion

Fig. 1. Effect of H2O2 on biomass (a) and photosynthetic parameters (b) of two lettuce genotypes. Different letters indicate significant difference
at P < 0.05. * and ** indicate significant differences between treatment with P < 0.05 and P < 0.01, respectiveely. Pn, net photosynthetic rate;
Gs, stomatal conductance; Tr, transpiration rate; WUE, water use efficiency. The 5 to 20 mmol/L H2O2 application increased the shoot and root
fresh weight, of which 10 mmol/L H2O2 addition showing the maximum improvement by 12.3% and 13.6% for Ziluoma, and 22.1% and 18.1%
for Lvluo, respectively
• 2nd Paper Results & discussion

Table 1. Effects of H2O2 on concentrations of individual phenolic
compound in two lettuce genotypes.
• 2nd paper Results & discussion
Except for chicoric acid 1, most of the identified phenolic compounds were
improved by H2O2 treatment in Ziluoma.

Fig. 2. Effect of H2O2 on the content of phenolic compounds and enzymes related to phenolic biosynthesis in lettuce plants. (a) Total phenolic, total flavonoid, and anthocyanin contents; (b)
Phenylalanine ammonia-lyase (PAL) activity; (c) Heat map showing the relative gene expression levels of enzymes involved in phenolic biosynthesis, including PAL, chalcone synthase (CHS),
flavanone 3-hydroxylase (F3H), dihydroflavonol-4-reductase (DFR), flavonoid 3′, 5′-hydroxylase (F35H), and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) in two lettuce genotypes;
(d) Critical enzymes in the phenolic biosynthesis pathways. * and ** indicate significant differences between treatment with P < 0.05 and P < 0.01, respectively. Compared with the control
treatment, the total phenolic content significantly enhanced by 51.3% and 20.0%. PAL activity significantly increased by 11.0% and 13.3% in Ziluoma and Lvluo, respectively, after H2O2
treatment. The significantly increases of most genes related to the phenolic biosynthetic pathway were observed after H2O2 treatment

Fig. 3. Effect of H2O2 on the content of (a) ascorbic acid (AsA), (b) reduced glutathione (GSH), (c) carotenoids, (d) soluble sugar, (e) soluble protein,
and (f) free amino acid in two lettuce genotypes. * and ** indicate significant differences between treatment with P < 0.05 and P < 0.01, respectively.
The higher concentrations of AsA, GSH, carotenoids, soluble sugar, soluble protein, and free amino acid were also observed in the H2O2-treated
lettuce, which enhanced by 23.6%, 137.9%, 22.2%, 19.5%, 13.3%, and 23.2%, respectively, in Ziluoma

Table 2. Effects of H2O2 on mineral contents in two genotypes
of lettuce leaves.
As compared with control treatment, the K, Ca, Mg, Fe, Cu, Zn, and B contents were significantly increased in both of two lettuce genotypes treated
with H2O2. the H2O2 treatment also significantly improved the S content by 33.2% in Ziluoma and P content by 19.6% in Lvluo, respectively

Fig. 4. Effect of H2O2 on antioxidant capacity (a) and the correlation of growth, photosynthetic parameters, quality parameters, and antioxidant activities in Ziluoma (b) and Lvluo (c). * and **
indicate significant differences between treatment with P < 0.05 and P < 0.01, respectively. Red and blue color represent positive and negative correlation, respectively. The large and small
circular sizes correspond to strong and weak correlations, respectively. SFW, shoot fresh weight; Total Chl, total chlorophyll content; Car, carotenoids content; Pn, net photosynthetic; TF, total
phenolic content; TF, total flavonoid content; TA, total anthocyanin content; AsA, ascorbic acid content; GSH, glutathione content; SS, soluble sugar content; SP, soluble protein content; FAA,
free amino acid content; MAN, macronutrient (P, K, Ca, and S) contents; MIN, micronutrient (Fe, Cu, Zn, and B) contents; The H2O2 treatment significantly improved the DPPH, FRAP, and
ABTS by 15.1%, 36.5%, and 30.1%, respectively, in Ziluoma, and by 4.1%, 9.1%, and 21.3%, respectively, in Lvluo

Conclusion
• All humanities benefits from the discovery of new foods. Elicitation has evolved to be one of the new approaches diversifying plant
metabolites and improving their functional properties
• There are various methods and strategies to improve plant foods via elicitation. However, ultrasound and chemical elicitation discussed in the
current study stand out as the most elicitation techniques
• These methods proved the efficacy to enhance secondary metabolites, primarily polyphenols in plant leaves and sprouts thereby providing an
effective way to improve health function of these plants.
• Using ultrasound, it was possible to increase functional compounds and biological activities of C. asiatica leave. Moreover, elicitation using
hydrogen peroxide showed a promising strategy in developing lettuce with high bioactive compounds and functional properties.

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