Universidad CES

Pharmacognosy
Midterm II - April 25th 2020

Given name/Surname: Javier Alejandro Daza Galván I.D.1065850412

Bonus: exam will be graded on an 85% (full marks) basis. However, you can not exclude any questions with a value of 5% or
higher. The highest possible grade is 5.0

1) Questions related to the chapter: “The Study of Plant Natural Product Biosynthesis in the Pre-genomics and Genomics Eras”

a) (5%) Organize the following activities if you are pretending to use a hypothetical biochemical approach to study natural product
biosynthesis:

i) Heterologous Protein expression.

ii) Peptide Micro-sequencing.
iii) Partial cDNA Cloning by PCR.
iv) Identification of enzymatic reaction.
v) Full-Length cDNA Cloning.
vi) Enzyme purification.
vii) Enzyme activity confirmation.

Order: 1)iv, Identification of enzymatic reaction 2) vi, enzyme purification, 3)ii, peptide micro-sequencing, 4) iii, partial cDNA cloning by
PCR, 5) v, full length cDNA cloning, 6) I, Heterologous protein expression and 7) vii, enzyme activity confirmation

b) (5%) Explain the differences between a Forward-Genetic approach and a reverse-genetic approach when studying gene function.
Answer: In Forward-Genetic approach the altered phenotype first obtained by induce mutation in genes, in this one we want to know
what is the mutant that caused this altered phenotype.

In reverse-genetic approach, the phenotype is the last one thing obtained, it means, that we must have to identify which is the gene
that altered the phenotype to identify gene function.

c) (2.5%) Select the best description about how TT8 gene function was studied in Arabidopsis thaliana.
i) TT8 was studied using a forward-genetic approach. This gene is involved in the synthesis of Flavonoids through phenylpropanoid
pathway.
ii) TT8 was studied using a forward-genetic approach. This gene is involved in the synthesis of alkaloids through shikimic acid
pathway.
iii) TT8 was studied using metabolomics. This gene is involved in the synthesis of tannins through phenylpropanoid pathway.
iv) TT8 was studied using homology-based molecular approach. This gene was involved in the synthesis of polyketides through
malonic acid pathway.
v) TT8 was studied using a transcriptomic approach. This gene is involved in the synthesis of tannins through phenylpropanoid
pathway.

d) (10%) DNA sequences of several Taxus species transferases were aligned using the Clustal W algorithm on the MultAlin Web site
(http://prodes.toulouse.inra.fr/multalin/multalin.html) and the BoxShade service to color the nucleotides
(www.ch.embnet.org/software/BOX_form.html). Letters in black boxes indicate identical base pairs as compared to the first sequence.
Letters in gray boxes indicate similar base pairs. The sequences include: Taxus cuspidata 10-deacetylbaccatin III-10-O-acetyl transferase
(DBAT) mRNA; Taxus baccata 10-deacetylbaccatin III-10-O-acetyl transferase (DBAT) mRNA; Taxus cuspidata taxadienol acetyl
transferase (TAT) mRNA; Taxus cuspidata taxoid-O-acetyltransferase (TOAT) mRNA; Taxus cuspidate phenylpropanoyltransferase
(BAPT) mRNA; and Taxus media phenylpropanoyltransferase (BAPT) mRNA.
 i) Using homology-based molecular approach: which enzyme shares the highest similitude sequence with Taxus cuspidata 10-
deacetylbaccatin III-10-O-acetyl transferase (DBAT)?
Answer: The enzyme which shares the highest similitude sequences with DBAT is T. baccata DBAT

ii) Are the transferases genes with the highest structural similitude homologous or orthologous?
They are homologous, because orthologous is about the same function no matters gene similitude.

iii) A homology-based molecular approach must be used in the case of convergent gene evolution?
Answer: No, because the enzyme of different plants species evolved despite that perfomed a same biochemal functions, it means
that the gene sequence changes due to sequences variations by this evolution

e) (2.5%) Which of the following techniques it is NOT a platform frequently used in proteomics?
i) two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).
ii) serial analysis of gene expression (SAGE).
iii) mass spectrometry (MS).
iv) yeast two-hybrid array systems.
v) protein arrays (using antibodies as probes).

f) (2.5%) Which of the following techniques is, today (2020), the best option if you want to do large-scale metabolic profiling in a
metabolomics study?
i) gas chromatography (GC)/MS.
ii) Liquid chromatography/MS (LC/MS).
iii) nuclear magnetic resonance (NMR) spectroscopy.
iv) Liquid chromatography/nuclear magnetic resonance (LC/NMR).
v) Liquid chromatography/Fourier-transform ion cyclotron mass spectrometry (LC/FTMS).

g) (10%) Answer the following questions based on the paper by A. Aharoni et al., “Identification of the SAAT gene involved in strawberry
flavor biogenesis by use of DNA microarrays,” Plant Cell, 2000.

According to the Strawberry and Petunia cDNA microarrays,

i. Describe what the white boxed cDNA clone shown in panel B

could mean?
That cDNA clone is very preserve
ii. Identify 2 Strawberry cDNA clones (row and column) that
could be associated with SAAT gene.
1°cDNA clon : row -> 2, column ->3
2°cDNA clon : row->4, column-> 1
iii. Identify 2 cDNA Strawberry clones (row and column)
preferentially expressed in ripen Strawberries.
- Row ->4, column->2
- Row ->1 , column ->3
iv. Identify 2 cDNA Strawberry clones (row and column)
preferentially expressed in unripe Strawberries.

v. Identify 2 cDNA Strawberry clones (row and column)

expressed at the same level in either ripen and unripe
fruits.

Figure. Strawberry and Petunia cDNA Microarray

(A) The strawberry and petunia cDNAs were spotted in a 4 x 4 format using a 16-pin print head. In each of the 16 subarrays, the first
12 columns from the left are strawberry probes (total of 1701, in duplicate), and the four columns from the right are petunia probes
(total of 480, in duplicate). The array area is 17 x 17 mm. The image is a two-color overlay obtained with green-stage target
(fluorescently labeled with Cy5) and red-stage target (fluorescently labeled with Cy3) cohybridized with a single microarray. In the
2) Questions related to the chapter: “bioassays for activity”

a) (2.5%) Extracts of following species have been found to have g) (2.5%) Mammalian primary tissue cell cultures are frequently
antiviral activity against Herpes simplex: used in the evaluation of natural products to:

i) Acnistus arborescens from Costa Rica. i) Test anticancer compounds.

ii) Atropa Belladonna from France. ii) Determine cyto-toxicity and genotoxicity in "healthy" cells.
iii) Trichilia Roka from the United States. iii) Calculate lethal dosis in humans.
iv) Camptotheca acuminata from China. iv) Test antibacterial activity.
v) Cannabis sativa from India. v) Calculate the dose of the natural compound to be delivered
in animal patients.
b) (2.5%) The inhibition assay of cathepsin K and falcipain-2 allows
to search for compounds with: h) (2.5%) The figure shown below shows the results after the
incubation of erythromycin in a microbroth dilution assay of a
i) Antibacterial and antifungical activity. second isolated strain of Escherichia coli. The dark wells denote
ii) Anthelmintic and anticancer activity. growth, the empty wells (in white) denote absence of growth.
iii) Antimalarial and anti-histaminic activity. After incubation, the contents of each well were grown in a non-
iv) Anti-osteoporosis and antifungical activity. selective medium. Which of the following is the best
v) Anti-osteoporosis and anti-malaria activity. interpretation of the results?

c) (2.5%) Trypanothione reductase inhibition assays are useful for

discovering compounds with possible activity to treat:

i) Chagas disease and “sleeping sickness”.

ii) Osteporosis.
i) The minimum inhibitory concentration is 1 mg / ml and the
iii) Prostate cancer.
drug is bacteriostatic.
iv) Malaria.
ii) The minimum inhibitory concentration is 1 mg / ml and the
v) Zika.
drug is bactericidal.
iii) The minimum inhibitory concentration is 2 mg / ml and the
d) (2.5%) The following biological activity assays are based on gel
drug is bacteriostatic.
electrophoresis.
iv) The minimum inhibitory concentration is 2 mg / ml and the
drug is bactericidal.
i) Inhibition of topoisomerase II.
v) The minimum inhibitory concentration is greater than 1 mg
ii) Inhibition of papain, cathepsin and falcipain.
/ ml and the drug is bactericidal.
iii) Anti Herpes simplex virus.
iv) Trypanothione reductase inhibition assay.
i) (2.5%) Azadirachtin and Nomiline are substances with:
v) Inhibition of tubulin polymerization.
i) Bacteriostatic, fungicidal and antineoplastic activities.
e) (2.5%) Which of the following is an advantage of the microbroth
ii) Antineoplastic, antifeedant and molluscicide activities.
dilution assay?
iii) Deterrent, insecticide and growth disruptor activities.
iv) Anthelmintic, antifeedant and antifungal activities
i) It can be used to determine antibiotic resistance and its
v) Cytotoxic, Antineoplastic and antibacterial.
susceptibility.
ii) It produces results faster than the other methods.
j) (2.5%) What types of bioassays can be carried out with Epilachia
iii) It does not require isolation of a micro-organism.
varivestis Muls (Mexican bean beetle)?
iv) It is more accurate than the other methods.
v) It can be used to determine the minimum bactericidal
i) Double-choice anti-feedant assay.
concentration of the drug.
ii) Anti cancer assays.
iii) Anti-Herpex simplex virus assays.
f) (2.5%) The diagram shown below shows the results after
iv) Nematocidal assay.
incubation of a pure compound obtained from plants tested
v) Cytotoxicity assays.
using microbroth dilution assay with an isolate of Escherichia
coli. The dark wells denote growth, the empty wells (in white)
k) (2.5%) The image on the left shows
denote absence of growth. After incubation the contents of each
a sensitivity test using the disk
well were grown in a non-selective medium. Which of the
diffusion method. In this case, it is
following is the best interpretation of the results?
shown the effect of different
antibacterial compounds on a
bacterium. All antibiotics are of
natural origin except "A1" which
represents a chemically modified
version of "A". Based on these
i) The minimum inhibitory concentration is 0.25 mg / ml and results, what can be concluded
the drug is bacteriostatic. about antibiotics "A" and "A1"?
ii) The minimum inhibitory concentration is 0.25 mg / ml and
the drug is bactericidal. i) The modification increases the minimum inhibitory
iii) The minimum inhibitory concentration is 0.5 mg / ml and concentration (MIC) of "A1".
the drug is bacteriostatic. ii) The modification decreases the MIC of "A1".
iv) The minimum inhibitory concentration is 0.5 mg / ml and iii) The modification increases the lethal concentration 50 of
the drug is bactericidal. "A1".
v) The minimum inhibitory concentration is 1 mg / ml and the iv) The modification gives a broader spectrum of action to
drug is bactericidal. "A1".
v) The modification has no effect on the MIC of "A1".
 l) (2.5%) The following microbial strain can be used to test n) (2.5%) Tigenone, is used a positive control in:
compounds for ANTIFUNGAL activity via microbroth dilution
assay i) Mammary cancer in vitro assay.
ii) Prostate cancer in vitro assay.
i) Bacillus cereus. iii) Liver cancer in vitro assay.
ii) Pseudomonas aeruginosa. iv) Prostate cancer in vitro assay.
iii) Escherichia coli. iv) Bladder cancer in vitro assay.
iv) Staphylococcus aureus
v) Saccharomyces cerevisiae. o) (2.5%) Which of the following compounds have anthelmitic
properties?
m) (2.5%) Plant extracts for microbroth dilution assay are usually
dissolved in dimethyl sulfoxide (DMSO) because: i) Santonin.
ii) N,N-diethyl-m-toluamide (DEET).
i) DMSO does not affect microbial grow at any concentration. iii) SEVIN (1-naphthyl N-methylcarbamate).
ii) DMSO has antibacterial activity. iv) Nomilin.
iii) DMSO helps to suspend low polar substances into high v) Formazan.
polar solvents.
iv) DMSO has a strong antifungical activity. p) (2.5%) Mollusks can be vectors in the transmission of:
iv) DMSO is an antibacterial synergist compound.
i) Herpes simplex virus.
ii) Chagas disease.
iii) Schistosomiasis.
iv) Dengue virus.
v) Zika virus.

3) Questions related to the chapter: “Traditional, Analytical, and Preparative Separations of Natural Products”

a) (2.5%) After collecting a plant species for the extraction of d) (2.5%) In a normal phase thin layer chromatography (TLC)
metabolites, the most advisable is to: experiment, two compounds show retention factors (Rf) of 0.89
and 0.93. How should the experiment be modified to improve
i) Dry it at low temperature until water is extracted from the the separation?
tissues.
ii) Freeze it in liquid nitrogen and then store it in the fridge at i) Use less solvent in the mobile phase.
-80°C. ii) Use more solvent in the mobile phase.
iii) Dry it at high temperatures quickly. iii) Allow the mobile phase solvent to move further on the top
iv) Grind it immediately and let it dry. of the plate.
v) Keep the specimen in high humidity to preserve it better. iv) Allow the mobile phase solvent to move less on the top of
the plate.
v) Use a more polar solvent in the mobile phase.

b) (2.5%) Paper chromatography is a separation technique that can

be use to purify natural products that:
e) (2.5%) What active compounds can not be extracted using
i) Have low polarities and low molecular weights (<300 Da). microwave-assisted extractions:
ii) Have high polarities and high molecular weights (> 300 Da).
iii) Separates molecules by hydrophobic interactions. i) Taxanes from Taxus spp.
iv) Only serves to separate high molecular weight compounds ii) Cocaine from Erythroxylon coca.
like chlorophylls and carotenoids. iii) Artemisin from Artemisa annua.
v) Can not be used in the separation of molecules of natural iv) Amrphous silica gel from Equisetum arvense.
products. v) Ginsenoside from Panax ginseng.

f) (2.5%) Which of the following substances inhibit topoisomerase

c) (2.5%) Supercritical CO2 fluid is a good option to extract: II?
i) Bark tannins of Hypericum perforatum.
ii) Alkaloids from Papaver somniferum. i) Lupeol.
iii) Essential oils from Lavandula angustifolia. ii) Linalool.
iv) Caffeic acid from Coffea Arabica. iii) Camphor.
v) Lignin from Abbies grandis. iv) Artemisin.
v) Praziquantel.

g) Consider the following compounds and answer the following questions related to thin layer chromatography (TLC):

a. (3%) Order the compounds from lower retention

factor (Rf) to higher Rf if we ran a normal
phase TLC:
Answer:
1° A , 2°C and 3° B
b. (4.5%) A TLC plate is spotted with samples of A, B and C. a TLC is ran using as mobile phase a mix of Hexane:Ethanol (5:95). The
figure shows the results after TLC run. ¿How could you change the mobile phase to
achieve a better compound separation?
Answer: Decreasing the ethanol concentration, and adding some of hexane but, keeping more ethanol concentration until to see a good separation
resolution