Laboratory Exercises Ver20220909

UNIVERS ITY OF COPENHAGEN
FACULTY OF HEALTH AND MEDICAL SCIENCES
Drugs from Nature

Laboratory exercise manual
Copyright 2022
Department of Drug Design and Pharmacology

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Preface
On behalf of the course team, I welcome you to the laboratory exercises for 'Drugs from Nature' (in
Danish 'Lægemidler fra Naturen'). The course covers the curriculum within the research area Natural
Products Research/Pharmacognosy, which is mandatory for pharmacy students – first of all because
Pharmacists sell standardized herbal products from pharmacy stores, but also because bioactive
natural products play an important role in drug discovery and development.
We have developed six 4-hours exercises that each gives you as course participant practical
experience with methods used for analysis of herbal products according to the European
Pharmacopeia (Ph. Eur.) as well as advanced methods used for studying bioactive constituents in
natural sources such as plants and microbial extracts. We hope these exercises will stimulate your
curiosity towards bioactive constituents in complex mixtures, and also provide you a deeper insight
and understanding of the methods used in Natural Products Research.
This manual for the laboratory exercises is written in English because many of the teachers are
English speaking. Likewise, short reports for each exercise must be written in English. This gives you
the opportunity to practice your English within the field of Natural products research, but feel also
free to discuss words and terminologies with the Danish-speaking teachers throughout the course.
This is the fourth year we provide Drugs from Nature, and we have incorporated changes based on
our experience with the laboratory exercises last year. Still, we very much appreciate feedback to all
exercises immediately after you have conducted the exercises. You can do this in the 'Laboratory
exercise evaluation book' that is available in the teaching laboratories.
Copenhagen – August 2022
Professor Dan Stærk

Natural Products Research group
Department of Drug Design and Pharmacology
University of Copenhagen
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Contents
PREFACE ............................................................................................................................... 3
PRACTICAL INFORMATION ................................................................................................. 8
Laboratory safety ............................................................................................................................................................... 8
Groups and overview of exercises ..................................................................................................................................... 8
22 OBLIGATORISKE SIKKERHEDSSPØRGSMÅL .......................................................... 10
EXERCISE 1 – BOTANICAL EXCURSION AND HERBAL DRUGS ................................. 15

Aim..................................................................................................................................................................................... 15
Preparation ....................................................................................................................................................................... 15
Schedule and meeting points ........................................................................................................................................... 15
Droge-øvelse på NaturMedicinsk Museum .................................................................................................................... 16
Spørgsmåls- og svarark til droge-øvelse på NaturMedicinsk Museum ...................................................................... 18
EXERCISE 2 – PURPLE CONEFLOWER HERB ............................................................... 20

Background ....................................................................................................................................................................... 20
Preparation for exercise 4................................................................................................................................................ 21
Aim..................................................................................................................................................................................... 21
Preparation ....................................................................................................................................................................... 21
Experimental..................................................................................................................................................................... 22
Corrections and comments to PURPLE CONEFLOWER HERB .............................................................................. 26
Data analysis and report writing .................................................................................................................................... 27
Post-lab checklist Exercise 2............................................................................................................................................ 28
Post-lab checklist Exercise 4.1.1 (week 37) .................................................................................................................... 29
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EXERCISE 3 – GINKGO DRY EXTRACT ........................................................................... 30
Background ....................................................................................................................................................................... 30
Preparation for exercise 4................................................................................................................................................ 31
Aim..................................................................................................................................................................................... 31
Preparation ....................................................................................................................................................................... 31
Experimental..................................................................................................................................................................... 31
Corrections and comments to GINKGO DRY EXTRACT, REFINED AND QUANTIFIED ................................. 35
Data analysis and report scheme .................................................................................................................................... 36
Post-lab checklist Exercise 4.1.2 (Week 38) ................................................................................................................... 38
Post-lab checklist Exercise 4.2.1 (Week 38) ................................................................................................................... 38
Report scheme for exercise 3 ........................................................................................................................................... 39
EXERCISE 4 – CHARACTERISATION OF FUNGAL CULTURES ................................... 41
Sources of filamentous fungi ........................................................................................................................................... 41
Secondary metabolite potential ....................................................................................................................................... 41
Identification of filamentous fungi.................................................................................................................................. 42
Aim..................................................................................................................................................................................... 42
Preparation ....................................................................................................................................................................... 43
EXERCISE 4.1: ISOLATION OF ENDOPHYTIC FUNGI .................................................... 43
Exercise 4.1.1: Isolation of endophytic fungi – WEEK 37............................................................................................ 44
Materials for exercise 4.1.1: Isolation of endophytic fungi – WEEK 37 ..................................................................... 44
Experimental for exercise 4.1.1: Isolation of endophytic fungi – WEEK 37 .............................................................. 44
Exercise 4.1.2: Isolation of endophytic fungi – WEEK 38 and 39 ............................................................................... 45

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Experimental for exercise 4.1.2: Isolation of endophytic fungi – WEEK 38 and 39.................................................. 46
EXERCISE 4.2: CHEMICAL INVESTIGATION OF AN ENDOPHYTIC FUNGUS ............ 46

Exercise 4.2.1: Chemical investigation of an endophytic fungus – WEEK 38............................................................ 47
Materials for exercise 4.2.1: Chemical investigation of an endophytic fungus – WEEK 38 ..................................... 47
Experimental for exercise 4.2.1: Chemical investigations of an endophytic fungus – WEEK 38............................. 48
Exercise 4.2.2: Chemical investigation of an endophytic fungus – WEEK 39............................................................ 49
Materials for exercise 4.2.2: Chemical investigation of an endophytic fungus – WEEK 39 ...................................... 49
Experimental for exercise 4.2.2: Chemical investigations of an endophytic fungus – WEEK 39............................. 49
EXERCISE 4.3: MORPHOLOGICAL INVESTIGATION OF FILAMENTOUS FUNGI ....... 51
Materials for exercise 4.3 – WEEK 39: Morphological investigations of filamentous fungi .................................... 52
Experimental for exercise 4.3 – WEEK 39: Morphological investigations of filamentous fungi ............................. 52
Data analysis and report writing for exercises 4.1-4.3.................................................................................................. 54
Post-lab checklist Exercise 4.1.2, 4.2.2 and 4.3 (Week 39)............................................................................................ 55
EXERCISE 5 – ISOLATION OF LOVASTATIN FROM RED YEAST RICE ...................... 56

Background ....................................................................................................................................................................... 56
Aim..................................................................................................................................................................................... 58
Preparation ....................................................................................................................................................................... 58
Experimental..................................................................................................................................................................... 58
Material ............................................................................................................................................................................. 58
Part A: Preparation of crude red yeast rice extract ..................................................................................................... 59
Part B: Isolation of lovastatin by flash chromatography ............................................................................................. 59
Part C: NMR spectroscopic analysis and structure determination of sample with isolated lovastatin ................... 62
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Part D: Mass spectrometric analysis of sample with isolated lovastatin .................................................................... 62
Data analysis and report scheme .................................................................................................................................... 63
EXERCISE 6 – PTP1B INHIBITORS IN EREMOPHILA SP. .............................................. 65

Time and place for mandatory exercise ......................................................................................................................... 65
Background ....................................................................................................................................................................... 65
Preparation for theoretical exercise 6 ............................................................................................................................ 66
Aim..................................................................................................................................................................................... 66
Experimental data available ............................................................................................................................................ 66
Tasks for exercise 6 .......................................................................................................................................................... 67

Task 1 - Construction of a biochromatogram ................................................................................................................ 67
Task 2 - Determination of IC50 value ............................................................................................................................ 67
Task 3 - Analysis of HRMS .......................................................................................................................................... 68
Task 4 - Analysis of NMR data ..................................................................................................................................... 68
Report scheme for exercise 6 ........................................................................................................................................... 72
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Practical information
Below, you will find a brief description of laboratory safety and organisation of the laboratory
exercises
Laboratory safety
Laboratory safety is important whenever you work in a laboratory – not only for yourself, but also
for your fellow students and the teachers. This is especially important in the teaching laboratories
where many people are working at the same time on a relatively limited space. However, by following
laboratory safety-rules and by behaving carefully and sensible, it is possible to minimize risk factors
and ensure a safe working environment. A safety manual for work in laboratories at University of
Copenhagen can be found in Danish and in English on Absalon. You must have read and understood
these before starting the laboratory exercises. Thus, before the first day in the laboratory you must
have answered (and passed) the assignment on Absalon with safety-related questions. You will
not be allowed to start the laboratory exercises without passing the safety question assignment.
There will also be a brief safety demonstration by the instructors at the first day of the laboratory
exercise.
Groups and overview of exercises

Exercises are placed Tuesday to Thursday in the afternoon, and the teams (in Danish 'Hold') are
mostly distributed on these days according to the below scheme (laboratory exercise teachers are
indicated in brackets after the teams). There will be two teachers and one lab-technician to assist in
the laboratories.
NOTE: Laboratory exercises start at 12:30 and ends at 16:30
The laboratory exercises are performed in groups of two students, and you must sign up in these
groups via Absalon before the laboratory exercises start. Please start from the lowest group numbers
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to avoid empty groups in between.We of course expect you to have prepared yourself by reading the
laboratory exercise before showing up in the laboratory, and as part of this you must have passed the
‘pre-lab assignment’ given as assignments for exercise 2 - 6. Nobody is allowed to start the
laboratory exercises without passing the pre-lab exercise assignments. Both members of the
groups are of course also expected to work actively during the exercise and to discuss and interpret
results. Likewise, both group members should participate in preparing a short written exercise report
(in English if you have an English-speaking supervisor at the laboratory course). At the end of the
day, you must fill out a post-lab checklist that the teachers must approve before you leave the
laboratory.
Data from HPLC analyses, LC-HRMS analyses and NMR analyses will typically be available on
Absalon one or two days after the exercises. The report should be handed in one week after the
laboratory exercise was conducted. Laboratory reports will be evaluated with passed/not passed – and
upon active participation in the laboratory exercises and having the laboratory exercises passed, the
participants will obtain a course certificate with the evaluation passed. This is a prerequisite for taking
the 2-hour written exam after the course.
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22 obligatoriske sikkerhedsspørgsmål
Nedenstående sikkerhedsspørgsmål skal besvares online som assignment i Absalon før
laboratorieøvelserne starter. Der kræves minimum 17 point for at få adgang til laboratoriet.
Bemærk at der kan være flere korrekte svar per spørgsmål og at alle korrekte svar skal angives.
1. Hvad gør du hvis der sker en alvorlig ulykke med en tilskadekommet person i laboratoriet?
 Få fat i en underviser
 Yd førstehjælp
 Tilkald politiet, ring 114
 Tilkald ambulance, ring 112
 Informer Campus Service SUND
2. Hvor findes den nærmeste hjertestarter?

 Det kan mobil-App’en ’Hjertestart’ fortælle mig
 Bygning 30, balkonrum, stueetage
 Bygning 11, ved elevator, stueetage, vest
 Bygning 22, indgang til kantine
 Bygning 10, trapperepos, 1. etage
3. Hvad gør du ved en forgiftningsulykke?

 Ring evt. til kemisk beredskab eller giftlinjen for vejledning
 Tag den forgiftede til egen læge
 Ring 1813 og få den forgiftede til en akutmodtagelse
 Ledsager af den forgiftede bør evt. medbringe forskrifter, brugsanvisninger el.lign.
4. Hvordan transporterer du en kolbe, med en opløsning der skal inddampes, til

rotationsfordamperen?
 Forseglet med staniol
 Forseglet med prop
 Forseglet med papirsserviet
 Forseglet med egen hånd
 Mærket med indhold
 Mærket med dato, holdnummer og gruppenummer
 Mærket med kemisk formel
5. Hvem tømmer forlaget på rotationsfordamperen efter brug?

 Laboranten
 Jeg beder kursusvejleder om dette hvis jeg ikke selv har tid
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 Mig selv - den velopdragne studerende
 Den tilstedeværende underviser
6. Hvor befinder øjenskyllere sig normalt i et kemilaboratorium?

 Ved vaskene
 I min taske
 Ved døre
 Ophængt på væggen
 Ophængt udenfor laboratoriet
 Ved stinkskabe
7. Hvad gør du hvis der går ild i et elektrisk apparatur?

 Slukker for strømmen hvis muligt
 Slukker ilden med vand
 Slukker ilden med tørt sand
 Slukker ilden med pulver-slukker
 Slukker ilden med kulsyre/CO2
 Slukker ilden med tæppe/kvælning
8. Hvorfor skal kitlen altid være knappet, og hvorfor bruger vi altid sikkerhedsbriller i et
kemilaboratorium?
 For at sikre at vi ikke biologisk kontaminerer vores prøver
 For at skåne vores almindelige tøj
 For at beskytte os mod brandskader, derfor er kitlen lavet af bomuld
 For at beskytte os mod brandskader, derfor er kitlen lavet af kunststof
 For at beskytte os mod indtag af skadelige stoffer
 For at beskytte hud og øjne mod skadelige stoffer og/eller splintrende effekter
9. Hvordan vil du afmåle koncentrerede syrer og baser?

 Altid iført handsker
 Overføres fra original emballage til min flaske med målepipette
 Overføres fra original emballage via mindre beholder til min flaske
 Ved hjælp af mundpipettering
 Jeg beder en laborant gøre det da jeg ikke selv tør
10. Hvorfor skal stinkskabslugen være lukket ned til under øjenhøjde, når der arbejdes der?
 For at sikre at jeg kan stikke overkroppen ind under lugen
 For at beskytte mit ansigt mod de kemiske stoffer jeg arbejder med
 For at beskytte mine kolleger i lokalet mod de kemiske stoffer jeg arbejder med
 For at sikre tilstrækkeligt sug i stinkskabet
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AFFALDSHÅNDTERING.
11. Hvilken affaldsgruppe skal diethylether (C2H5)2O hældes i?
O
K
Z
T
X
A
B
C
H
12. Hvilken affaldsgruppe skal dichlormetan CH2Cl2 hældes i?

O
K
Z
T
X
A
B
C
H
13. Hvilken affaldsgruppe skal ethanol CH3CH2OH hældes i?

O
K
Z
T
X
A
B
C
H
 Jeg hælder det i vasken
 Jeg drikker det
14. Må man sidde på laboratoriebordet?

 Ja, vi har mangel på stole grundet de årlige 2-procents besparelser
 Ja, hvis du ikke kan se når underviseren forklarer
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 Nej, så bliver man sendt hjem
 Nej
15. Hører TLC-plader ind under kemikalieforurenet glasaffald?

 Nej, de skal blot i affaldsgruppe H dunk
 Ja, de skal i ikke-klassificeret affaldsspand med glas
 Nej, de skal blot i affaldsgruppe C dunk
 Ja, de skal i bøtten i stinkskab markeret affaldsgruppe H
 Nej, de skal blot i skraldespanden
16. Når kolben skal tages af rotationsfordamperen, skal vakuum så slippes før eller efter dette
påbegyndes, og hvordan er stinkskabslugen placeret?
 Stinkskabslugen skal være trukket ned til under hovedhøjde
 Stinkskabslugen skal være fuldt oplukket
 Vakuum slippes hurtigt før kolben tages af
 Vakuum slippes forsigtigt inden kolben tages af
 Kolben tages af og vakuum slippes forsigtigt
 Jeg beder en laborant gøre det for mig
17. Hvad gør du, hvis du eller en medstuderende får kemikalier i øjene?
 Fjerner kontaktlinser
 Anbefaler min medstuderende at vente til det går over
 Skyller øjnene med øjenbruseren
 Skyller øjnene med øjenskylleflasker
 Skyller øjnene med svag base hvis der er tale om syre i øjnene
18. Hvor længe skal TLC-plader afdampe, før de kan tages ud fra stinkskabet?
 Mindst 5 min
 Mindst 30 min
 Den behøver ikke afdampe
 Når den ikke lugter af organisk solvent længere
19. Skal man have handsker på til alt arbejde i et kemilaboratorium?

 Ja, altid
 Ja, når jeg står i nærheden af et stinkskab
 Nej, kun ved arbejde med syrer og baser
 Nej, kun når der er risiko for at få farlige kemikalier på hænderne
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20. Når du har brugt glasudstyr til organiske opløsningsmidler, skal det så vaskes op med det
samme?
 Ja, det er vigtigt straks at sætte glasudstyr til opvask
 Nej, det er ikke nødvendigt
 Nej, det skal afdampe i stinkskab først
21. Hvad tjekker du ved din arbejdsplads i laboratoriet før du går hjem?
 At du har fået skrevet en hilsen med sprittusch til laboranterne
 At der er ryddet op
 At posen i affaldsbøtten i stinkskabet er tømt over i de blå affaldscontainere og ny pose sat
i
 At lugen er efterladt helt åben for at sikre udluftning
 At spild er fjernet
 At alt flydende affald er hældt i affaldsdunk C
22. Hvis brandalarmen går, hvad gør du så?

 Færdiggør hurtigt mit laboratoriearbejde og forlader så laboratoriet
 Samler mine ting sammen og forlader laboratoriet
 Forlader straks laboratoriet
 Går direkte til samlepladsen
 Går hjem
 Tager elevatoren til stueplan
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Exercise 1 – Botanical excursion and herbal drugs
Plants continue to be an important source of bioactive chemical entities for treatment of many
diseases. This is both as i) phytotherapy by traditional medicine practitioners in many third world
countries, where the healers collect local medicinal plants based on generations of orally inherited
knowledge, ii) as part of written traditional medicinal systems like the traditional Chinese medicine
(TCM), iii) as standardized herbal products prepared according to various pharmacopeias and sold in
pharmacies worldwide, and finally iv) as sources of new bioactive chemical entities in drug discovery.
No matter the use, knowledge of plant taxonomy, plant morphology and other properties of plants are
important for identification and collection of plant material. This exercise consists of an excursion to
the Botanical Garden and a small exercise on herbal drugs in 'Naturmedicinsk Museum'.
Aim
The aim of the botanical excursion and the herbal drug exercise in 'NaturMedicinsk Museum' is to
provide students with a brief inspirational introduction to the historical as well as current use of
medicinal plants – and start discussions of subjects such as plant morphology, plant taxonomy,
traditional use, current use, standardization of herbal remedies, and plants as sources of new chemical
entities in drug discovery.
Preparation
Before the botanical excursions, participants should read Chapter 3 and 4 in the textbook. The youtube
channel “Botanik for farmaceuter” containing 25 videos is also recommended as a useful introduction
to the subject, but note that these videos are in Danish and use Danish terminology
(https://www.youtube.com/playlist?list=PL9y77XXcZAjNk15K90pFGn5wnuN-Bke1Y ).
For the excursion, be prepared for any kind of weather – check the weather forecast and bring
umbrella/rain coat if needed, and sensible shoes. Do not expect to be able to take notes on laptops or
tablets in the botanical garden, but bring your laptop to the museum.
Schedule and meeting points

The meeting point for the botanical excursion is in front of the Botanical Garden Shop, just inside the
garden at the gate to Nørreport Station (Gothersgade 130, 1123 København K). Don’t be late, as we
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will quickly disappear into the Garden! The meeting point for the herbal drug exercise at
NaturMedicinsk Museum is on the 3rd floor of building 22.
Each team will visit the Botanical Garden as well as carry out the herbal drug exercise in
'NaturMedicinsk Museum' on the same day. We have reserved minimum 30 min between the two
activities, which should be enough time for transport from Universitetsparken to the Botanical Garden
(or vice versa). You will be responsible for transport forth and back between the Botanical Garden
and NaturMedicinsk Museum (NMM) at Universitetsparken according to the schedule below:
Team 1: Tuesday September 6th 12:30-14:30 Botanical Garden and 15:00-16:30 NMM
Team 2: Tuesday September 6th 12:30-14:00 NMM and 14:30-16:30 Botanical Garden
Team 3: Wednesday September 7th 12:30-14:30 Botanical Garden and 15:00-16:30 NMM
Team 4: Wednesday September 7th 12:30-14:00 NMM and 14:30-16:30 Botanical Garden
Team 5: Thursday September 8th 12:30-14:30 Botanical Garden and 15:00-16:30 NMM
Team 6: Thursday September 8th 12:30-14:00 NMM and 14:30-16:30 Botanical Garden
Droge-øvelse på NaturMedicinsk Museum

Øvelsens formål er træning af drogekendskab og planteklassifikation (Heinrich kap. 3), samt brug
af relevante internet-ressourcer hvor denne viden kan findes. Øvelsen kræver adgang til internettet
og adgang til Ph. Eur. 10.0 eller nyere (Ph. Eur. Online: https://pheur.edqm.eu/home). Der er et
begrænset antal licenser til Ph. Eur. Online, så derfor skal én repræsentant for hver gruppe selv
registrere sig for at få en licens som beskrevet på bibliotekets hjemmeside:
https://kub.kb.dk/sund/farma
I vil kort blive introduceret til NaturMedicinsk Museum. Herefter trækker hver to-mands gruppe en
seddel med et engelsk navn på en droge som findes i Ph. Eur 10.0, hvorefter nedenfor anførte
opgaver løses:
• Opgave 1: Find drogen på museet og tag et billede af den
• Opgave 2: Brug museet og de listede internet-ressourcer til at besvare spørgsmålene i
nedenstående svarark som kan downloades som Word-version fra Absalon.
Det udfyldte svarark udgør en øvelsesrapport for Exercise 1 og skal uploades til godkendelse af
rapporten på Absalon. Afleveringsfristen er 2 hverdage efter øvelsen.
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Når opgaverne er løst
Brug eventuelt den resterende tid på museet til at studere drogetyper (radix, rhizhoma, herba,
folium, etc) og Ph. Eur droger. Museet indeholder også historiske lægemiddelplanter, hvoraf
mange er ophav til moderne, syntetiserede lægemidler, samt bioaktive planter som bruges i alt fra
folkemedicin til rusmidler.
Nyttige hjemmesider
• Ph. Eur online: https://pheur.edqm.eu/home. Kræver log-in og adgang gennem REX og/eller
Eduroam.
• Plants of the World Online: http://www.plantsoftheworldonline.org. Den bedste og mest
opdaterede liste over plantearter, deres klassifikation, distribution mm)
• European Medicines Agency: https://www.ema.europa.eu/en. Lægemiddelagenturet, udgiver
bl.a. drogemonografier som indeholder terapeutiske indikationer, bivirkninger, interaktioner, mv.
For nogen droger findes også lister for i hvilke lande drogen indgår i godkendte produkter.
• Lægemiddelstyrelsen: https://laegemiddelstyrelsen.dk. Lægemiddelstyrelsen udgiver Danske
Lægemiddelstandarder (DLS) som sætter bestemmelserne i Ph. Eur. i kraft i Danmark, og i
nogen tilfælde også supplerer dem. Lægemiddelstyrelsen udgiver bl.a. også en opdateret liste
med navne på godkendte naturlægemidler i DK, og danske navne for Ph. Eur. Droger.
• Google Scholar, Scopus, PubMed og lignende.
• Google og Wikipedia er dine venner, men husk at være kildekritisk.
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Spørgsmåls- og svarark til droge-øvelse på NaturMedicinsk Museum
Find drogen på museet, tag et billede og indsæt det herunder:

Indsæt billede her:
Find den latinske titel på drogens Ph. Eur. monografi.

Besvarelse:
Find plantens gældende binominelle navn (”latinske navn”). Hvis drogen kan stamme fra flere arter,
så vælg én - gælder også for følgende spørgsmål.
Besvarelse:
Hvilken plantefamilie tilhører arten?

Besvarelse:
Har arten tidligere været placeret i andre slægter (jf. dens synonymer som kan findes i POWO)?
Besvarelse:
Hvad er artens danske navn?

Besvarelse:
Vokser arten i Danmark?

Besvarelse:
Indgår drogen i et eller flere godkendte naturlægemidler i Danmark? Hvis ja .angives præparatnavn
Besvarelse:
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Har European Medicines Agency (EMA) udgivet en endelig (”final”) monografi for drogen?
Besvarelse:
Er drogens terapeutiske indikationer i EMA-monografien (uanset evt publiceringsstatus) angivet

under ”veletableret anvendelse”, ”traditionel anvendelse” eller begge?
Besvarelse:
Skriv en reference til mindst én peer-reviewed videnskabelig artikel som understøtter en eller flere
af indikationerne angivet af EMA
Besvarelse:
Find og tag billeder af tre andre typer droger som alle er optaget i Ph. Eur. (med drogetype menes
droge baseret på en anden plantedel).
Indsæt billeder her:
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Exercise 2 – Purple coneflower herb
Background
Purple coneflower herb (Echinacea purpurea
herba) is used in traditional medicine in North
America and Europe to treat a wide variety of
conditions, from snakebites to bacterial and viral
infections, and its preparations are one of the
most important and popular herbal products on
the market.1 However, the immunomodulatory
effect of herbal preparations of purple Figure 2.1. Echinaceae pupurea. Source: Kindscher, K. in
coneflower herb is recognised as the main reason Echinacea: Herbal Medicine with a Wild History. Springer.
2016. p. 150
of its use, since it can help in the treatment and
prevention of the common cold, influenza and other upper respiratory tract infections. Clinical studies
report significant efficacy and safety of the pressed juice from fresh flowering purple coneflower on
reducing the duration of the common cold in humans and significant immunomodulatory effects are
also reported in rat immune systems. 2
The most abundant components of Echinacea purpurea herba are alkamides, glycoproteins,
polysaccharides, and phenolic compounds derived from caffeic acid: caffeoyl glycosides (e.g.
echinacoside, 1), caffeoyl quinic acids (e.g. chlorogenic acid, 2), and caffeoyl tartaric acids (e.g.
caftaric acid, 3 and cichoric acid, 4). Even though not conclusive yet, studies report the activity of
preparations of purple coneflower as due to a synergism of the main constituents. 3
1
Barnes J, Anderson LA, Gibbons S, Phillipson JD. Echinacea species (Echinacea angustifolia (DC.) Hell., Echinacea
pallida (Nutt.) Nutt., Echinacea purpurea (L.) Moench): a review of their chemistry, pharmacology and clinical
properties. Journal of Pharmacy and Pharmacology 2005, 57, 929-954
2 Sculten B, Bulitta M, Ballering-Brühl B, Köster U, Schäfer M. Efficacy of Echinacea purpurea in patients with a
common cold. Drug Research 2001, 51, 563-568.
3 Kindscher, K. Echinecea: Herbal medicine with a wild history. Springer. Switzerland. 2016.
20
Figure 2.2. Structures of major constituents found in Echinacea purpurea.
Preparation for exercise 4

'Exercise 4.1.1: Isolation of endophytic fungi – WEEK 37' should be carried out today. Thus, all
uneven groups (1, 3, and 5) should START performing exercise 4.1.1 FOLLOWED by exercise 2.
All even groups (2, 4, and 6) should START performing exercise 2 FOLLOWED by exercise 4.1.1.
Aim
The aim of this exercise is to identify the presence of the major compounds caftaric acid and cichoric
acid in the dried plant material of purple coneflower herb and certify if the plant material is in
accordance with the European Pharmacopoeia (Ph. Eur.).
Preparation
As always, we expect you to come well-prepared to the laboratory exercises. Thus, in addition to
having read this exercise (as well as the relevant parts of exercise 4), you must have conducted the
‘Prelab exercises’ available as an assignment on Absalon. Furthermore, you must have seen the below
demonstration videos available on this YouTube playlist: https://tinyurl.com/DrugsFromNature
• Video 1 - How to do TLC
• Video 4 - Isolation of endophytes
21
Experimental
Conduct the exercise in accordance with the below Ph. Eur. monograph PURPLE CONEFLOWER
HERB - but take into account all corrections and comments indicated with red notes in the monograph
and provided in the bullet list after the monograph.
22
Note 5
Note 1
Note 2
Note 3
Note 4
Note 5
Note 6
Note 7
23
Note 9
Note 10
Note 11
Note 8
24
25
Corrections and comments to PURPLE CONEFLOWER HERB
IDENTIFICATION:
• Note 1 - Identification: Only C and D are performed in this exercise.
• Note 2 - Reference: Reference solution is provided.
• Note 3 - TLC plate: TLC plates of 5 × 7.5 cm are provided.
• Note 4 - Mobile phase: Prepare 10 mL in a 50 or 100 mL conical flask, add constituents in the
order listed, mix gently, and transfer approximately 7 mL to the TLC chamber. IMPORTANT:
The TLC solvent should not cover the spotted samples. Always keep the lid on the chamber to
avoid evaporation of the mobile phase.
• Note 5 - Application: Spot reference and test solutions (do not add bands as stated) on a line
approximately 1 cm from the bottom of the TLC plate. Use capillary tubes to spot 25 µL of both
reference and test solution (0.5 cm capillary height equals 5 µL, put a finger on the upper tube
end to make smaller spots), let the spots dry before applying more solution and make sure the
spots are completely dry before placing the plate in the TLC chamber. When placing the plate
in the TLC chamber, ensure the spots are not submerged into the liquid. Let the mobile phase run
until 0.5 cm from the top of the TLC plate (10-20 min).
• Note 6 - Drying: Let the TLC plate dry for 10 minutes in your own fume hood.
• Note 7 - Detection: Firstly, examine the dried TLC plate under UV light at both 254 and 365 nm.
Secondly, heat up the TLC plate 1 minute using the heat gun in the TLC station (without
excessive temperatures), and dip the TLC plate in the detection solution available in the TLC
station while the TLC plate is still hot. Leave the plate to dry in the marked beakers in the TLC
station for 15-20 minutes, before finally examining it again under UV light at 365 nm.
TESTS:
• Note 8: Skip the 'Total ash' experiment, but make sure to start Loss on Drying at the beginning
of the exercise day!!! Weigh the cruicible (small ceramic container) without lid.
• ASSAY (Liquid Chromatography):
• Note 9 - Sample for HPLC analysis: Notice that this is not the same extract as for the TLC
analysis. Prepare a sample (approximately 1 mL) for HPLC analysis by filtering the extract
through a 0.45 m syringe filter. Place the HPLC vial (properly marked with team number and
group number) in the autosampler tray placed next to the HPLC system. You can find the correct
26
position of your vial in the column named 'vial' next to your Team/group number in the column
named 'Sample name'.
• Note 10 - Reference solution: A reference solution of chlorogenic acid and caffeic acid has been
prepared by the course team, and HPLC analysis of this sample will be performed by the course
team.
• Note 11 - Column and mobile phase: The HPLC system is set up with the correct mobile phase,
column, and a sample table with the proper HPLC analysis for all groups.
Data analysis and report writing

The HPLC data from the ASSAY will be available on Absalon approximately 24 hours after the
laboratory exercise – if no major instrumental breakdown occurs. The data should be analysed
according to the pharmacopeia, vide supra. All other data should be collected (write down raw data
like tare- and gross weights from weighing, start and end measurements from TLC, photos, etc) by
yourself during the exercise.
The report (max 3-4 pages + appendices) should contain the following
1. Aim (short and concise)
2. Background (where you briefly elaborate on the traditional and current use, effect, and chemical
constituents in Echinacea purpurea beyond the text in this laboratory manual)
3. Experimental (special emphasis on modifications compared to the pharmacopeia)
4. Results and discussion (highlighting the main findings according to IDENTIFICATION, TESTS
and ASSAY from the pharmacopeia)
5. Conclusion (short and concise)
6. The reports should be uploaded to the assignment named 'Report exercise 2' with deadline
Friday September 23rd.
27
Post-lab checklist Exercise 2
We have…
 Photograph(s) of the TLC plate after successful development
 Prepared an HPLC vial with the test solution, labelled it with team and group number, and
placed it by the HPLC
 Data from loss on drying
Does the TEST loss on drying comply with the Ph. Eur. requirements?
 No, because __________________________________________
 Yes, because __________________________________________
Has the TLC plate eluted properly, i.e., narrow bands/spots, no tailing, etc?
 No, because __________________________________________
 Yes, because __________________________________________
Does IDENTIFICATION by TLC comply with the Ph. Eur. requirements?

 No, because __________________________________________
 Yes, because __________________________________________
How will you calculate the relative retention for peaks in the HPLC chromatogram?
Which two formulas can you use to calculate resolution (system suitability) for the HPLC
chromatogram? How do you decide which one to use?
28
We have…
 Cleaned up our fume hood and washed all used glassware
 Emptied waste containers (fume hood and bench) and put new waste bags in the containers
Write the formulas to calculate the percentage of caftaric acid and cichoric acid from Ph. Eur.
below.
Describe what A1, A2, and A3 are, and calculate C1 and C2

A1:
A2:
A3:
C1:
C2:
Post-lab checklist Exercise 4.1.1 (week 37)
We have…
 Noted experimental procedures, plant genus and species, habitat, and GPS coordinates for
our collected plants
 Noted how long we surface sterilized our plant material
 Uniquely labelled our petri dishes with HxGy and media on the bottom periphery and HxGy
on the side of the lid
 Securely sealed our petri dishes and placed them next to the incubator
Post-lab checklist approved by teacher
Signature teacher
29
Exercise 3 – Ginkgo dry extract
Background
Ginkgo biloba, also known as the temple tree, has been used in
traditional Chinese herbal medicine since 1504 AD, but was not
introduced to Europe till the 1960s.4 Herbal medicine containing
standardized extracts of Gingko biloba is used to alleviate a broad
range of disorders, including cognitive dysfunctions, headache,
tinnitus, vertigo, inattention, mood disturbances, cardiovascular
diseases, and coronary heart disease.5 Certain pharmacological
properties of the Gingko biloba extracts have been demonstrated in animal and human studies,
including antioxidative activities, increase of tolerance to hypoxia, and improvement of blood
rheology by increasing the flexibility of cellular blood components, thus enhancing microcirculation,
affecting neurotransmitter levels, enhancing neuroplasticity, prevention of brain edema, and
neuroprotection.6 Ginkgo biloba contains flavonoids, terpene lactones, and ginkgolic acids. Although
sub-fractions of the extracts show some activity in pharmacological models, it does not generally
reproduce the actions of the total extract5 - and it is therefore likely that additive, antagonistic and/or
synergistic effects of the different active constituents occur at various molecular target sites in
different organs and tissues.5
Figure 3.1. Structures of chlorogenic acid and selected flavonoids found in Ginkgo biloba.
4
Isah T. Rethinking Ginkgo biloba L.: Medicinal uses and conservation. Pharmacognosy Reviews 2015, 9, 140-148.
5
DeFeudis F-V, Drieu K. Ginkgo biloba extract (EGb 761) and CNS functions: basic studies and clinical applications.
Current Drug Targets 2000, 1, 25-58.
6
Zhang H-F, Huang L-B, Zhong Y-B, Zhou Q-H, Wang H-L, Zheng G-Q, Lin Y. An overview of systematic reviews of
Ginkgo biloba extracts for mild cognitive impairment and dementia. Frontiers in Aging Neuroscience 2016, 8, 276.
30
Preparation for exercise 4
'Exercise 4.2.1: Chemical investigation of an endophytic fungus – WEEK 38' and 'Exercise 4.1.2:
Isolation of endophytic fungi WEEK 38 and 39' should be carried out today. Thus, all uneven groups
(1, 3, and 5) should START performing exercise 4.1.2 and 4.2.1 followed by exercise 3. All even
groups (2, 4, and 6) should START performing exercise 3 followed by exercise 4.1.2 and 4.2.1.
Aim
The aim of this exercise is to become familiar with herbal medicine monographs in the European
Pharmacopeia (Ph. Eur.) – specifically a monograph for refined herbal extracts – and to communicate
the results from the conducted experiments in a report.
Preparation
• Video 5 - Three-point inoculation of a fungus (first 43 seconds of video)
Experimental
Conduct the exercise in accordance with the below Ph. Eur. monograph GINKGO DRY EXTRACT,
REFINED AND QUANTIFIED - but take into account all corrections and comments indicated with
red notes in the monograph and provided in the bullet list after the monograph.
31
Note 7
Note 8
Note 9
Note 10
Note 11
Note 12
Note 13
Note 1
Note 2
Note 3
Note 4
Note 5
Note 6
32
33
34
Corrections and comments to GINKGO DRY EXTRACT, REFINED AND QUANTIFIED
IDENTIFICATION by thin-layer chromatography (TLC)
• Note 1 - A reference solution containing chlorogenic acid and rutoside is provided.
• Note 2 - TLC plate: 7 x 4 cm plates are provided.
• Note 3 - Mobile phase: Prepare 10 mL in a 50 or 100 mL conical flasks (be careful with water
into acid!), mix gently, and transfer only approximately 7 mL to the TLC chamber.
IMPORTANT: The TLC solvent should be well below the area with the spotted samples. Always
keep the lid on chamber to avoid evaporation of the mobile phase.
• Note 4 - Application: Spot reference and test solutions on a line 1 cm from the bottom of the
TLC plate. Use capillary tubes to spot 5 µL solution (0.5 cm capillary height equals 5 µL), let
the spots dry before repeating this three times more for a total application of 20 µL solution. It
is very important that the spots are dry before inserting the plate in the TLC chamber.
When placing the plate in the TLC chamber, ensure the spots are not submerged into the liquid.
Let the mobile phase run until it is approximately 0.5 cm from the top of the TLC plate (10-20
min).
• Note 5 - Drying: Leave the developed TLC plate to dry at room temperature in your fume hood
for 10 minutes.
• Note 6 - Detection: Firstly, examine the dried TLC plate under UV light at 365 nm. Secondly,
heat up the TLC plate 1-2 minutes using the heat gun in the TLC station (without excessive
temperatures). Thirdly, dip the TLC plate in detection solution 1 (diphenylboric acid aminoethyl
ester) inside the fume hood while the plate is still hot, and let it drip off for 30 seconds before
dipping in detection solution 2 (macrogol). Leave the plate to dry in the marked beakers in the
TLC station for 20 minutes, before finally examining it again under UV light at 365 nm.
ASSAY liquid chromatography (LC)
• Note 7 - Only perform analysis for flavonoids: The full Ph. Eur. monograph contains in addition
analysis for terpene lactones and ginkgolic acids.
• Note 8 – dissolution of ginkgo extract: Make sure that the extract is completely dissolved. If not,
sonicate the sample for a few minutes until everything is dissolved.
• Note 9 - Test solution: We use brown vials with a rubber membrane and a screw cap instead of
the aluminium crimp cap.
• Note 10 - Water-baths set to 90 °C is available for heating the sample.
35
• Note 11 - Sample for HPLC analysis: Prepare a sample (app 1 mL) for HPLC analysis by
filtering the cooled (room temperature) extract through a 0.45 m syringe filter. Place the HPLC
vial properly marked with team number and group number in the autosampler tray placed next
to the HPLC system. You can find the correct position of your vial in the column named 'vial'
next to your Team/group number in the column named 'Sample name'.
• Note 12 - Reference solution containing quercetin dihydrate has been prepared for all teams and
will be submitted to HPLC by the course-team.
• Note 13 - HPLC system: The HPLC system is set up with the correct mobile phase, column, and
a sample table with the proper HPLC analysis for all groups.
Data analysis and report scheme

The HPLC data from the ASSAY will be available on Absalon approximately 24 hours after the
laboratory exercise – if no major instrumental breakdown occurs. The data should be analysed
according to the pharmacopeia, vide supra. All other data should be collected (write down raw data
like tare- and gross weights from weighing, start and end measurements from TLC, photos, etc) by
yourself during the exercise. The 'p-value', i.e., the 'purity-value' or the 'percentage content of
anhydrous quercetin in quercetin dihydrate CRS' is 81.7%
The below 'Report scheme' should be filled out and uploaded to the assignment named
'Report exercise 3' with deadline Friday October 30th.
36
We have…
 Photograph(s) of the TLC plate after successful development
 A test solution sample has been prepared in an HPLC vial, labelled with team and group
number, and placed by the HPLC (reference sample is provided)
Has the TLC plate eluted properly, i.e., narrow bands/spots, no tailing, etc?
 No, because __________________________________________
 Yes, because __________________________________________
Does IDENTIFICATION by TLC comply with the Ph. Eur. requirements?

 No, because __________________________________________
 Yes, because __________________________________________
How will you calculate the relative retention for peaks in the HPLC chromatogram?
How will you calculate the resolution for the HPLC chromatogram?
Why do you add diluted hydrochloric acid to the test solution?
Write the formula to calculate the percentage of flavonoids from Ph. Eur. below.
37
Describe what F1 and F2 are, and write the values of m1, m2 and p (remember units when relevant).
m1:
m2:
F1:
F2:
p:
We have…
 Emptied waste containers (hood and bench) and put new waste bags in the containers
Post-lab checklist Exercise 4.1.2 (Week 38)
We have…
 Obtained pictures of the petri dishes with (hopefully) endophytes from our collected leaf
material
Post-lab checklist Exercise 4.2.1 (Week 38)

We have…
 Correctly labelled all our petri dishes (HxGy and medium)
 Stacked our petri dishes and secured the stack with tape vertically (to make it easier to find
them next time)
 Visually inspected and photographed the endophytic fungi that we inoculated last week
 Wiped the table with 70% ethanol and cleaned up
Signature teacher
38
Report scheme for exercise 3
Insert picture(s) from your TLC analysis:
What does it show? Discuss the results according to the Ph. Eur. requirements.
Calculate the relative retentions for kaempferol and isorhamnetin (show the calculation and which
parameters you use) and comment on the result according to Ph. Eur. requirements:
Calculate the resolution for the HPLC chromatogram. Does it comply with Ph. Eur.? Why?
Calculate the percentage of flavonoids. Does it comply with Ph. Eur.?
39
Is the refined ginkgo dry extract in accordance with the Ph. Eur.? Explain your reasoning.
What would you do differently if you could re-do this lab exercise with the knowledge you have
now? Explain.
Evaluate the experimental error from your analysis. What are the main issues (if any)?
Insert your test and reference HPLC chromatograms:
40
Exercise 4 – Characterisation of fungal cultures
Filamentous fungi are a rich source of secondary metabolites of medical importance such as the tri-
peptide penicillin G (1), which revolutionized our ability to cure infections, and the polyketides
lovastatin (2) (cholesterol lowering agent) and mycophenolic acid (3) (immunosuppressant). Fungi
are, however, also a rich source of mycotoxins such as aflatoxin B1 (4) and patulin (5).
Sources of filamentous fungi

A traditional source of filamentous fungi is soil samples (terrestrial filamentous fungi) but other
promising sources of bioactive metabolites are marine fungi, isolated from e.g., marine sediments,
and endophytic fungi, which reside in the inter-cellular space within plants in a non-pathogenic
fashion. Due to fungi’s ability to adapt, the same fungal species isolated from soil, marine sediment
or plants can potentially produce a diverse range of metabolites.
Secondary metabolite potential

While filamentous fungi possess the genetic potential to produce a wide range of secondary
metabolites, the genes coding for these are often silenced under ‘standard’ laboratory conditions. One
way to express the silent genes and thus take advantage of the plasticity of fungi is to cultivate them
under different conditions such as media composition, temperature and cultivation near other
organisms (co-cultivation).
41
Identification of filamentous fungi
As is the case with plants, fungi can be identified to genus and species level. This can be done (by a
seasoned mycologist) through investigation of the macro- and micromorphology or by investigation
of specific gene sequences.
Note: As filamentous fungi need to grow in an incubator for approximately 7-14 days before
analysis this exercise will start already in week 37 according to the flowchart below:
Week 37 38 39
Exercise Isolate endo-

phytic fungi
Visually inspect
endophytes
Visually inspect
endophytes
4.1 4.1.1 (p 41-45) 4.1.2 (p 46) 4.1.2 (p 46)
Exercise Inoculate
fungus
Extract fungi
and investigate
4.2 4.2.1 (p 46-48) 4.2.2 (p 49-51)
Exercise Morphological
investigation
4.3 4.3 (p 51-54)
Aim
The aim of this exercise is three-fold:
• Familiarize yourself with the isolation procedures pertaining endophytic fungi and bacteria
(Exercise 4.1 – week 37 and week 39).
• Investigate the plasticity of filamentous fungi by chemical investigation at different
cultivation conditions (Exercise 4.2 – week 38 and week 39).
• Become acquainted with the macro- and micromorphology of fungi for identification at the
genus level (Exercise 4.3 – week 39)
42
Preparation
having read this exercise, you must have conducted the ‘Prelab exercises’ available as an assignment
on Absalon. Furthermore, you must have seen the below demonstration videos available on this
YouTube playlist: https://tinyurl.com/DrugsFromNature
• Video 4 - Isolation of endophytes
• Video 5 - Three-point inoculation of a fungus (plug extraction from 43 second)
• Video 6 - How to prepare a fungal microscopy slide
Exercise 4.1: Isolation of endophytic fungi

Endophytes, broadly described by Bacon et al. 7 as “microbes that colonize living, internal tissues of
plants without causing any immediate, overt negative effects”, are relatively unstudied as potential
sources of novel natural products for exploitation in medicine, agriculture, and industry. According
to one of the pioneers in endophyte research, Prof. Gary A. Strobel, of the approximately 300,000
higher plant species that exist on the earth, each individual plant, of the billions that exist here, is host
to one or more endophytes. 8 With conservative estimates of at least 1 million species of endophytic
fungi,9 these provide an important source of genetic diversity. With only a fraction of the world’s
plants having been fully studied relative to their endophytic biology, the opportunity to find new and
interesting endophytic microorganisms among myriads of plants in different settings and ecosystems
is promising.
EXERCISE 4.1 RUNS FROM WEEK 37-39: IN WEEK 37 WE WILL START THE
EXTRACTION OF ENDOPHYTES FROM THE COLLECTED LEAVES TO ALLOW THEM
TO INCUBATE FOR 14 DAYS UNTIL WEEK 39.
7
Bacon, C. W.; White, J. F. Microbial Endophytes, Marcel-Dekker: New York, 2000.
8
Strobel, G.; Daisy, B.; Castillo, U.; Harper, J. Natural products from endophytic microorganisms. J. Nat. Prod. 2004,
67, 257-268.
9
Dreyfuss, M. M.; Chapela, I. H. In: The Discovery of Natural Products with Therapeutic Potential; Gullo, V. P., Ed.;
Butterworth-Heinemann: Boston, 1994, pp 49-80.
43
Exercise 4.1.1: Isolation of endophytic fungi – WEEK 37
In this exercise, we will investigate different plant hosts exclusively for their associated fungal
endophytes by placing surface sterilized leaf plant material on growth media containing antibiotics.
Here we are cultivating the endophytes on plates with antibiotics, which are thus selective for fungi.
Materials for exercise 4.1.1: Isolation of endophytic fungi – WEEK 37

Each group should collect fresh plant material (leaves from two different plants) in a brown paper
bag no more than 48 hours before the exercises in week 37. Let the leaves air-dry a bit before
placing it in the bag. Bring it to the laboratory exercises. Note the GPS coordinates using your
smartphone, take photos of the host plants, and identify them using a plant identification app.
Recommended apps are iNaturalist or PlantSnap for iPhone (the latter restricted to 5 pictures per
day) or Google Lens or PictureThis for Android (again the latter with restricted number of free
pictures).
In addition to this, the following should be used:
• 0.01% Tween20
• 70% Ethanol
• 0.5% Sodium hypochlorite
• Sterile water
• Two petri dishes with potato dextrose agar (PDA) containing 50 ppm streptomycin and
tetracycline (inhibits bacteria).
• Empty petri dish
• Scalpel
• Tweezers (two)
• Parafilm
• Permanent marker
Experimental for exercise 4.1.1: Isolation of endophytic fungi – WEEK 37

The procedure is explained below in bullet points and illustrated in Figure 4.2:
1. Prepare the two different petri dishes by writing group number (i.e. H1G13 for group 13 on team
1) and type of agar medium along the border of the bottom of the petri dishes (see Figure 4.1).
2. Surface sterilize the table, tweezers, and scalpel with 70% ethanol.
44
3. Each leaf is submerged and washed in 0.01% Tween20 (15 seconds).
4. The washed leaves are surface sterilized by sequentially submerging them in 70% ethanol (30
sec), 0.5% sodium hypochlorite (1 min), and 70% ethanol (30 sec).
5. From each leaf, cut four pieces of approximately 20  2 mm in a petri dish using a sterile scalpel.
6. Each piece of plant material is submerged in 70% ethanol (10 seconds) followed by washing in
sterile water (5 seconds) using a sterilized tweezer. Place four pieces of the first leaf on a PDA
plate by gently lifting the lid to avoid contamination by airborne fungi – do not remove the lid.
Repeat this procedure, and place four pieces of the second leaf on the other PDA plate.
7. The two plates are wrapped together with parafilm (not tape) as shown in Figure 4.2 and placed
on the table close to where you found them. The teachers will cultivate them at 30 °C until week
39
Figure 4.1. Petri dishes should be marked with group and team numbers on the bottom of the plate as well as on the side of the lid
Figure 4.2. Workflow for isolation of endophytes from leaves. After surface sterilization, the leaf is cut into 20  2 mm pieces under
sterile conditions and subsequently sterilized again before placing two pieces in each petri dish. Wrap the petri dishes with parafilm
(not tape)
Exercise 4.1.2: Isolation of endophytic fungi – WEEK 38 and 39

Given that the surface-sterilization in week 37 was successful, and that the plant material contains
endophytes, your petri dishes should contain fungi with different macro-morphology.
45
Experimental for exercise 4.1.2: Isolation of endophytic fungi – WEEK 38 and 39
IMPORTANT SAFETY INFORMATIONS: When isolating fungi and bacteria the identities are
unknown until further microscopic and/or genetic investigations. Until we know the identity of the
microorganisms we must treat them as potential human pathogens. Thus, you must under no
circumstance open the lid of the petri dishes!
Do a macroscopic investigation of the incubated plates (after 7 and 14 days of incubation) and write
down the observed macroscopic features (colours of front and back, indication of growth rate, density
of mycelia, etc) as well as counting the number of fungi with different macro-morphology from the
two leaves. At the ENDOPHYTE PHOTO STATION: Take photos of the microplates from above
as well as with the bottom turned upside. If there is too much moisture on the lid for you to take the
required photos, contact a teacher for assistance. You must under no circumstance open the lid
of the petri dishes!
The report should contain the following

• Number of different colonies with reported macroscopic features (colours of front and reverse,
density of mycelia)
• Information of the source of the endophytes (plant genus and species, habitat, and GPS
coordinates)
• Photos of the petri dishes (from the top as well as from the bottom)
Exercise 4.2: Chemical investigation of an endophytic

fungus
Microorganisms such as filamentous fungi hold a huge genetic potential and have an impressive
ability to incorporate media components into biosynthetic pathways. Chemical metabolic profiling of
fungi has, however, shown low expression of the potential pathways and to be able to tap into the
gene pool of filamentous fungi and trigger the activation of silent genes, several approaches have
46
been proposed. These include genetic modification such as overexpression of promoters 10 and co-
cultivation with other microorganisms to promote a metabolic response. 11 A simple, yet effective,
approach is to activate silent genes through changing the cultivation parameters, including media
composition, light/dark cycles, temperature and ventilation. 12
In this exercise, you will three-point inoculate an endophytic fungus isolated in Nationalpark Mols
Bjerge, Denmark, on a range of cultivation media. After cultivation, you will observe the effect on
the phenotype of the inoculated fungi (exercises in week 39) and identify the genus based on
microscopic investigations. To investigate the effect on the metabolic profile, the fungal biomass will
be extracted with organic solvents and analysed by TLC for preliminary assessment of the metabolic
profile. Furthermore, HPLC-HRMS analysis of the extracts will form the basis for a subsequent
dereplication as part of the class exercises on October 6th.
Exercise 4.2.1: Chemical investigation of an endophytic fungus – WEEK 38

To investigate the chemical profiles of the endophytic fungus, we use the three-point inoculation
strategy, which allows us to extract mycelium in the confrontation zone with the other colonies as
well as the centre of the colony away from other colonies. You inoculate the fungus during the
exercises in week 38 to allow the colonies to grow for 7 days before extraction in week 39.
Materials for exercise 4.2.1: Chemical investigation of an endophytic fungus –

WEEK 38
• Three petri dishes with different growth media (see table below with media)
• Fungal biomass of an endophytic fungus in 60 mm petri dish
• Tape
10
Bergmann, S.; Funk, A. N.; Scherlach, K.; Volker, S.; Shelest, E.; Horn, U.; Hertweck, C.; Brakhage, A. A.
Activation of a Silent Fungal Polyketide Biosynthesis Pathway through Regulatory Cross Talk with a Cryptic
Nonribosomal Peptide Synthetase Gene Cluster. Appl. Environ. Microbiol. 2010, 76(24), 8143-8149.
11
Bertrand, S.; Schumpp, O.; Bohni, N.; Monod, M.; Gindro, K.; Wolfender, J.-L. De Novo Production of Metabolites
by Fungal Co-culture of Trichophyton rubrum and Bionectria ochroleuca. J. Nat. Prod. 2013, 76, 1157-1165.
12
Bode, H. B.; Bethe, B.; Höfs, R.; Zeeck, A. Big Effects from Small Changes: Possible Ways to Explore Nature's
Chemical Diversity. ChemBioChem, 2002, 3, 619-627.
47
• Sterile tooth pick
Media # Media
1 CYA: Czapek yeast agar
2 MEA: Malt Extract Agar
3 PDA: Potato Dextrose Agar
Experimental for exercise 4.2.1: Chemical investigations of an endophytic fungus –

WEEK 38
The procedure is explained below in bullet points and illustrated in Figure 4.3:
1. Collect the three petri dishes with different media according to the above table, and write media
code and group number (e.g., H1G13 for group 13 on team 1) along the border of the bottom of
the plates and on the side of the lid (see also Figure 4.1).
2. Use gloves for the remainder of this exercise!!
3. Place each petri dish to be inoculated upside down on the table without opening them. This is to
minimize contamination from airborne spores.
4. Gently lift the lid to the fungus and scrape off a small amount of spores and mycelia with an
inoculation needle or a tooth pick. By gently lifting a petri dish (still upside-down) make a three-
point inoculation with the inoculation needle (see Figure 4.3). Note that you are not streaking
out the fungus like you would with bacteria.
5. Repeat the procedure for the two other growth media. Dispose the inoculation needle if there is
risk of contamination.
6. The five petri dishes should be wrapped together with tape (not parafilm) as shown in Figure 4.2
and placed in the incubator for cultivation at 30 °C until week 39. Fungi like to grow mycelia
down. This will also ensure that the spores are on the fungus and not in the lid.
Figure 4.3. Workflow for three-point inoculation. From a spore suspension or fungal culture, three-point inoculations are done using
a sterile inoculation needle or a tooth pick.
48
Exercise 4.2.2: Chemical investigation of an endophytic fungus – WEEK 39
Uneven groups should start with 'Chemical investigations of an endophytic fungus' (this section:
Exercise 4.2.2 – week 39) in week 39 and start 'Morphological investigations of filamentous fungi'
(Exercise 4.3) after approximately 2 hours
Materials for exercise 4.2.2: Chemical investigation of an endophytic fungus –

WEEK 39
• Rack for Eppendorf tubes

• 6 Eppendorf tubes
• Plug drill
• Ethyl acetate + 1% formic acid (HPLC grade)
• 1000 µL Pipette
• Pasteur pipettes + balls
• Tooth pick
• TLC plate
• Capillary tubes
• TLC vessel
• TLC solvents (toluene:ethyl acetate: formic acid [6:3:1])
Experimental for exercise 4.2.2: Chemical investigations of an endophytic fungus –

WEEK 39
IMPORTANT SAFETY INFORMATIONS:
• As many fungal cultures contain toxic compounds, including carcinogenic substances (known
and unknown), all extracts should be considered toxic, gloves should be worn throughout
the exercise, and the work should be performed in the fume hood.
• If you spill extract on the table or racks, clean twice with 70% ethanol and dry with paper towel.
If your gloves are contaminated, change them immediately as they will only withstand
organic solvents for a few seconds.
49
• All plates and other consumables, which have been in contact with the fungus, should be
collected in a yellow plastic container and discarded as biological waste. Pipette tips and
Eppendorf tubes used for extracts are disposed of as ordinary laboratory waste.
Follow the procedure explained below in bullet points and illustrated in Figure 4.3:
1. Observe and take pictures of all fungi on different media for the report.
2. Label all Eppendorf tubes (on the lid) with
• Team number and group number (e.g. H1G13 for group 13 on team 1)
• Media code
3. Take the plug drill from the beaker with 70% ethanol. Using the plug drill, transfer three plugs
from a colony (see Figure 4.4) to an Eppendorf tube. Make the plugs at three different places,
i.e. at the center of a colony, at the edge of a colony close to the dish wall, and at the
confrontation zone between two colonies. Before making plugs from another medium, the plug
drill is cleaned twice in ethanol. Continue through the three media (in individual Eppendorf
tubes).
4. Using a 1000 µL single-use plastic pipette, add 500 µL ethyl acetate + 1% formic acid to each
of the three Eppendorf tubes and macerate the plugs extensively with a toothpick followed by
sonication for 30 min. The maceration is very important for an adequate yield.
5. After sonication, centrifuge the Eppendorf tubes for 5 min at max speed.
6. Transfer the supernatant to a clean Eppendorf tube marked with group number and media.
7. Carefully, with a capillary tube, spot 30 µL of each of the three extracts on a TLC plate.
8. Develop the TLC plate using a solvent system consisting of toluene:ethyl acetate:formic acid
(6:3:1)
9. Let the TLC plate dry in the fume hood for 10 minutes and observe it under UV light at 254
and 365 nm.
10. The remaining petri dishes should be put in white waste bags, emptied for air and closed
with a knot before they are discarded in the yellow bins for biological waste.
50
Figure 4.4. Workflow for plug extraction. Plugs are taken with a flame-sterilized cork borer throughout a single colony. The three
plugs are transferred to an Eppendorf tube for extraction. Remember to clean to cork borer when changing to a new media.

• Photos of the colonies on the three media and a macromorphological description of each of these
• Photos and discussion of the TLC plate observed under UV light at 254 and 365 nm
• Discussion about the chemical diversity of the fungus based on the TLC analysis.
Exercise 4.3: Morphological investigation of

filamentous fungi
Even groups should start with the 'Morphological investigation of filamentous fungi' (Exercise 4.3
– this section) in week 39 and start the 'Chemical investigation of an endophytic fungus (Exercise
4.2.2 – week 39) after approximately 2 hours
IMPORTANT SAFETY INFORMATIONS

Although the fungi we are working with in this exercise are not human pathogens it is important not
to inhale spores. Please work calmly when preparing microscopy slides and put the lid on the
petri dish when you are done. Wash your hands after handling the fungi.
When doing drug discovery from filamentous fungi it is helpful to know the identity of the fungus
under investigation. Finding a new fungus can increase the probability of finding novel chemistry
while the identification of a known fungus can assist the researcher in dereplication efforts as well as
help identify likely compound classes known from the genus. Gene sequencing of specific regions is
an effective method for identifying fungi to the species level, but an experienced mycologist can make
similar identifications by micro- and macroscopic investigations. In this exercise, you will try to
51
identify three fungi, one endophytic and two terrestrial, by preparing simple microscope slides and
investigating macroscopic features such as colours and growth. Each group should investigate three
different fungi, and petri dishes are shared between three groups (Groups 1-3, Groups 4-6, etc.). You
are welcome to share the slides but there should be enough mycelium for all groups to prepare their
own. Take pictures through the lens with your mobile phone, but you need a steady hand and a good
aim!
Materials for exercise 4.3 – WEEK 39: Morphological investigations of filamentous

fungi
At the MICROSCOPY SLIDE PREPARATION STATIONS:
• Three cultures for identification.
• Microscope slides in glass
• Mounting fluids (lactic acid and aniline blue)
• Scotch tape
At the MICROSCOPY STATIONS:
• Immersion oil
• Ethanol
• Lens paper
Experimental for exercise 4.3 – WEEK 39: Morphological investigations of

filamentous fungi
Follow the procedure explained below in bullet points and illustrated in Figure 4.5:
At the MICROSCOPY SLIDE PREPARATION STATIONS:
1. Conduct a macroscopic investigation of the fungi measuring colony size, colors (front and
reverse) and representation of mycelium.
2. Prepare two microscope slides by adding one drop of lactic acid as mounting fluid on one of the
slides and one drop of aniline blue as mounting fluid on the other slide
3. The colony is lightly touched with the adhesive side of scotch tape and transferred to a
preparations slide using a finger to slightly smoothen the tape on top of the slide. Note: try to
avoid excessive bubbles within the mounting fluid when mounting the scotch tape and avoid
getting mounting fluid on your fingers.
52
At the MICROSCOPY STATIONS:
4. Add a drop of immersion-oil on top of the tape, and investigate the slides with a light microscope
using 100× and 400× magnification.
5. Through microscopic investigation identify the morphological structures from the List of
morphological structures to identify below and use this to determine the genus of the three fungi.
6. Save microscope pictures of the micromorphological characteristics that you will use for
identification of your fungi.
Figure 4.5. Workflow for micro-morphological studies of filamentous fungi. Microscopic slides are prepared at the ‘Microscope
slide preparation stations’ using mounting fluid with or without dyes such as aniline blue. With the adhesive side of a piece of tape,
spores and mycelia are lifted from the colony (do not inhale spores) and transferred to a glass slide while removing excessive
bubbles. The microscopy slides are brought to the microscope area and a drop of immersion oil is added to allow microscopic
investigations at 100× and 400× magnification.
The report should contain

• Macroscopic observations
• Pictures, from the eyepiece cameras (or phone), of the morphological structures in the list below
with correct morphological terminology used.
• Discussion of the identity of the fungi based on your microscopic investigation – to the genus
level (you could also try to identify the species).
List of morphological structures to identify

• Identify a hypha – would you expect it to be septate or aseptate? What can you see?
• From each fungus, identify a conidiophore and describe it using correct terminology? (refer to
the lecture notes for inspiration)
53
• Identify the three fungi to the genus level using the fungal identification keys. If possible, try to
identify the species as well.
Data analysis and report writing for exercises 4.1-4.3

The TLC analyses should be discussed in the report as a chemical profiling. All data should be
collected (write down raw data, observations during microscopy, photos, etc) by yourself during the
exercise. Photos from the microscope cameras will be uploaded each day (use unique names for the
photos), but please try to obtain the photos yourself via USB during the exercise.
The report (max 4-6 pages + appendices) should contain the following
2. Background (where you describe endophytic fungi as a source of bioactive constituents and in
broad terms how they are isolated and characterized – and how their secondary metabolites are
extracted and identified)
3. Experimental (detailed information describing the work performed)
4. Results and discussion (highlighting the main findings – including the specific bulletpoints
mentioned under exercise 4.1-4.3)
5. Conclusion (short and concise)
6. The reports should be uploaded to the assignment named 'Report exercise 4' with deadline
Friday October 7th.
54
Post-lab checklist Exercise 4.1.2, 4.2.2 and 4.3 (Week 39)
4.1.2 (WEEK 39)

We have…
 Noted the macroscopic characteristics about the fungal colonies in each petri dish
 Obtained the necessary photo documentation for our report
4.2.2 (WEEK 39)

We have…
 Taken pictures of all fungi on different media, both front and reverse
 Obtained a meaningful result from our TLC analysis and remembered to photo-document it
 Observed chemical diversity from the TLC analysis
4.3 (WEEK 39)

We have…
 Measured the colony sizes of all three fungi
 Noted the front and reverse colors of the fungi
 Taken pictures of the macromorphology of all fungi
 Identified conidia and conidophores
 Taken pictures of micromorphological characteristics of all fungi
 Identified the genera of the three fungi
We have…
 Emptied waste containers in our fume hood and put a new waste bag in the container
 Wiped the table with 70% ethanol and cleaned up
Signature teacher
55
Exercise 5 – Isolation of lovastatin from Red Yeast Rice
This exercise is a slightly modified version of the exercise published in Nazri MM, Samat FD,
Kavanagh PV and Walsh JJ. J. Chem. Educ. 2012, 89, 138-140.
Background
Mevinolin or monacolin K, original names for lovastatin, has been isolated from Monascus ruber and
Aspergillus terreus. Apart from these fungal
species, Monascus purpureus, which is found
in traditional Chinese food containing red
yeast rice, has also proven to be a valuable
source of this compound.13 Clinical data
supports the use of red yeast rice for its Figure 5.1. Lovastatin in its lactam form and its ring-opened -
cholesterol lowering properties.14 Red yeast hydroxy acid form
rice or “Hongqu” (in Chinese) is prepared by fermenting a fungal strain, Monascus purpureus on
steamed rice which then produces lovastatin as one of its primary secondary metabolites. The
medicinal benefits of red yeast rice preparations have been recognized for thousands of years dating
back to 800 A.D. It is thought to enhance food digestion and blood circulation. Li Shizen, another
pharmacologist from the Ming Dynasty believed that “Hongqu” enhances “digestion and blood
circulation, can strengthen the spleen and dry the stomach”. Miao Xiyong, another pharmacologist
from the Ming dynasty also described “Hongqu”, according to the Ying system, as having significant
effect on the spleen and stomach. The secondary metabolites present in red yeast rice that are thought
to be responsible for its colour have been identified as monascin and ankaflavin (yellow),
monascorubin and rubropunctatin (orange), as well as monascorubramine and rubropuntamine
(red).15 Lovastatin, the active polyketide in red yeast rice,16 is not quite as colourful, but used
13
Ma, J.; Li, Y.; Ye, Q.; Li, J.; Hua, Y.; Ju, D.; Decheng, Z.; Cooper, R.; Chang, M. Constituents of Red Yeast Rice, a
traditional chinese food and medicine. J. Agric. Food Chem. 2000, 48, 5220-5225.
14
Heber, D.; Yip, I.; Ashley, J. M.; Elashoff, D. A.; Elashoff, R. M.; W Go, V. L; Cholesterol-lowering effects of a
proprietary Chinese red yeast rice dietary supplement. Am. J. Clin. Nutr. 1999, 69, 231-236
15
Pattanagul, P.; Pinthong, R.; Phianmongkhol, A.; Leksawasdi, N., Review of angkak production (Monascus
purpureus), Chiang Mai J. Sci. 2007, 34 (3), 319-328.
16
Murray, R. K.; Granner, D. K.; Mayes, P. A.; Rodwell, V. W. Harper's Illustrated Biochemistry Twenty Sixth
Edition; McGraw-Hill: United States of America, 2003; pp 205-211
56
clinically as a cholesterol-lowering drug for hyperlipidemia as well as for the treatment of obesity
and some cardiovascular diseases. There are also studies reporting that lovastatin has a desirable
effect on Alzheimer’s disease and in prevention of prostate cancer.17 Lovastatin, an inactive lactone
prodrug, is converted into its active form (β-hydroxy acid) (Figure 5.1) in the gastrointestinal tract
(GIT) by cytochrome enzymes P450 (CYP) 3A.18 When administered orally, it undergoes first-pass
metabolism through the liver causing considerable loss of its active metabolite(s), resulting in its low
bioavailability in blood. Consequently, the time taken to reach its peak concentration in blood is
longer (2 to 4 hours) than that of the other available statins. Like the other statins, lovastatin acts on
the HMG-CoA reductase enzyme which is involved in the first committed step in the de novo
synthesis of cholesterol in the liver. The β-hydroxy acid form of lovastatin resembles the structure of
HMG-CoA intermediate, which when reduced by HMG-CoA reductase produces mevalonate. It is
this enzyme that the active form of lovastatin competitively inhibits. With the cholesterol-lowering
effect of lovastatin, the level of low-density lipoprotein (LDL), “bad” cholesterol in the blood is
reduced while the level of high-density lipoprotein (HDL), “good” cholesterol, is increased gradually.
Reduced LDL levels in the blood is basically due to increased LDL uptake from the blood by the liver
and other peripheral tissues since less cholesterol is synthesized. Apart from this, the lower
cholesterol level in blood also reduces the conversion of HDL to its cholesterol esters (IDL and LDL)
thus increases HDL plasma level. These benefit the body since HDL functions to reduce deposition
of lipid and cholesterol in tissues, especially in vascular systems, hence the lowering effect of
lovastatin towards risk of developing any cardiovascular disorders.19 Lovastatin has short half-life of
approximately 3 hours in which it is eliminated by (CYP) 3A enzymes pathway in the liver.20 To
achieve maximal efficacy, it is therefore recommended that the drug is taken in the evening as there
is more synthesis of cholesterol at night as compared to during the day. Elimination of lovastatin from
the body is 83% via faecal excretion which includes from biliary systems and unabsorbed derivatives
17
Rang, P. H.; Dale, M. M.; Ritter, J. M.; Flower, R. J., Rang and Dale's Pharmacology 6th Ed.: Churchill-Livingstone
Elsevier: Philadelphia PA, 2007: pp 321-326.
18
Katzung, B. G. Basic and Clinical Pharmacology Ninth Ed.; McGraw-Hill Education (Asia): Singapore, 2004; pp
562-570.
19
Berg, J. M.; Tymoczko, J. L.; Stryer, L. Biochemistry Fifth Edition; W.H. Freeman and Company: Basingstoke,
England, 2002; pp 722-729.
20
Neuvonen, P. J.; Backman, J. T.; Niemi, M. Pharmacokinetic comparison of the potential over-the-counter statins
Simvastatin, Lovastatin, Fluvastatin and Pravastatin, Clin. Pharmacokinet. 2008, 47, 463-474.
57
while another 10% is eliminated via urine from the renal tubular system. Lovastatin is likely to
compete with other drugs, such as ranolazine (antianginal drug) and nimodipine (calcium channel
blocker), that also use (CYP) 3A enzymes in the GIT and liver for their metabolism resulting in
decreased metabolism of lovastatin as compared to when the other drugs are not present. Raised levels
of lovastatin can cause several mild undesirable effects such as myalgia, gastrointestinal disturbance,
insomnia, rashes and increased concentration of liver enzymes in plasma. The more severe yet rare
effects of lovastatin are rhabdomyolysis and angio-oedema. The drug is also contraindicated during
pregnancy since inhibition of HMG-CoA reductase may interrupt the natural migration of primordial
germ cells.
Aim
The aim of this exercise is to learn fast isolation of a natural product using a small-scale flash column
as well as molecular identification using mass spectrometry and NMR spectroscopy.
Preparation
• Video 2 - Packing and eluting a glass pipette column
• Video 3 - Preparing a NMR and MS sample
Experimental
Follow the procedures outlined in Part A-D below during this exercise, and interpret the spectral data
once they become available on Absalon.
Material
Red yeast rice used in this experiment is obtained from the dietary supplement preparation 'Rød ris'
from Natures Aid. Each capsule contains approximately 333 mg of red yeast rice and 10 mg of
monacolin K (lovastatin).
58
Part A: Preparation of crude red yeast rice extract
The content of four capsules of red yeast rice-containing dietary supplement should be weighed
directly in a tared Falcon tube. Add 5 mL of ethyl acetate and sonicate for 15 minutes, centrifuge for
2 minutes, and transfer the supernatant carefully to a 25 mL pear-shaped flask. Wash the remaining
material with 5 mL ethyl acetate, centrifuge again and transfer the supernatant to the pear-shaped
flask. Repeat the last washing step. Reduce the three combined supernatants to dryness using a rotary
evaporator and reconstitute in a minimum amount of dichloromethane to allow for easy transfer onto
the flash column. The lower the volume, the better chromatography – we are talking about a few
drops!
Part B: Isolation of lovastatin by flash chromatography

Background Information: The pioneer of flash
chromatography, W. C. Clark from Columbia University,
described this analytical tool as “basically an air pressure
driven hybrid of medium pressure and short column
chromatography which has been optimized for particularly
rapid separations”. Flash chromatography is essentially
normal phase column chromatography with silica gel as the
stationary phase. It boasts two fundamental differences
from conventional column chromatography. Firstly,
smaller silica gel particles (230-400 mesh) are employed.
Secondly, air pressure is applied to the column head to flush Figure 5.2. Schematics of a small flash column
the solvent through. The net result is a simple, rapid using a Pasteur pipette
chromatographic technique with high resolution power.
Column preparation: Ideally, a low viscosity chromatographic mobile phase should be selected
which separates the mixture and produces an Rf on TLC of approximately 0.24 for the desired
component. In this experiment, this has been done as part of the exercise development, which led to
a gradient mobile phase system, with different proportions of hexane to ethyl acetate changing from
2:1 to 1:1. Next, a column of appropriate diameter should be chosen. In this exercise, a standard
Pasteur pipette, with internal diameter of 5 mm, proves both an economical and effective approach.
59
Packing the column: Using a capillary tube or equivalent, insert a small plug of non-adsorbent cotton
wool into the end of a standard Pasteur pipette. This prevents the silica gel escaping from the column.
Next, add a 5-mm band of solid sodium chloride to yield a flat surface at the bottom of the column.
A plastic syringe without piston can be used as funnel (= 'syringe funnel'). Transfer silica gel (40-63
µm/230-400 mesh) to the column to a depth of 65 mm (using the same 'syringe-funnel' as described
above) in the fume hood, whilst gently tapping the column vertically on the bench to form an even,
compact column of gel that is about 55 mm in height. Spread a 5-mm layer of solid sodium chloride
over the top of the silica layer. When everything else is ready, solvate/equilibrate the silica gel
stationary phase by filling the column with hexane (approximately 2 mL or until all column material
is soaked in solvent and without air bubbles) using a Pasteur pipette. Do not allow the top of the
column to run dry at any time because this will permit re-entry of air into the system and ruin the
chromatography. Using a teat at the top of the column, push hexane through the column until any air
bubbles have escaped and the silica gel assumes a homogeneous appearance. If you let it ‘kiss’ the
top of the column when applying pressure, there is less risk of ruining the column than if you attach
the teat firmly to the Pasteur pipette - when you pull if off, you can easily create a vacuum that ruins
the column packing, so make sure to never suck from the top of the column. Remove the teat before
releasing the pressure.
Preparation of mobile phases: Prepare three mobile phases 1-3 as listed below in Falcon tubes and
make sure the lids are tightened to avoid evaporation:
Mobile phase 1: 1 mL of hexane
Mobile phase 2: 6 mL of hexane/ethyl acetate (2:1)
Mobile phase 3: 8 mL of hexane/ethyl acetate (1:1)
Sample application: When there is only a few millimetres of hexane above the silica gel, use a
Pasteur pipette to transfer the crude dichloromethane extract (from Part A) to the top of the
adsorbent bed. Rinse the flask with a few more drops of dichloromethane and add to the top of the
column. Let the extract sink into the stationary phase (apply a positive pressure to the column if
needed), and then perform a stepwise gradient elution using mobile phases 1-3, i.e., applying
aliquots of these on the top of the column in the indicated order, collect fractions of approximately
0.5 mL from the bottom of the column (mark corresponding height on sample tubes with a pen, so
you know when to change fractions). The fractions containing pure lovastatin are expected to elute
60
around fractions ~22-30, and this should be investigated by TLC of fractions ~15-30 (if all went as
planned) using silica gel TLC employing hexane/ethyl acetate (1:1) as mobile phase.
Identification of pure fraction(s): Spot, approximately 10 µL of each fraction for analysis, onto a
TLC plate of 10  20 cm dimensions. Also, apply 10 µL of the crude extract onto the plate as well
as 10 µL of pure lovastatin. Position the spots equidistant from each other and 2 cm up from the base
of the plate. Develop the plate and identify the lovastatin-containing fractions by visualizing under
UV light and by spraying with 1% vanillin reagent. Lovastatin visualizes as a blue-violet spot on a
yellowish purple background when the plate is sprayed with 1% vanillin/H2SO4 spray reagent when
left to dry in an oven for 10 minutes at 100 ºC. Lovastatin exhibits an Rf value of 0.24 when
hexane/ethyl acetate (1:1) is used as the mobile phase. Based on your analysis, pool fractions that
contain only pure lovastatin, i.e., those fractions in the beginning and the end that contain other
compounds should not be pooled with lovastatin. If you are in doubt, ask your teacher.
Conditions for TLC:

Stationary phase: Silica Gel GF254
Mobile phase: Hexane/ethyl acetate (1:1)
Plate size: 10 cm × 20 cm × 250 µm
Development height: 7.5 cm
Visualization: UV light at 254 and 365 nm followed by spraying with 1% vanillin
reagent and dry in oven at 100 C for 10 minutes*.
Reference solution: Lovastatin standard in acetonitrile
Test solutions: 30 fractions of eluent (~ 0.5 mL) collected from the column
*Vanilin reagent (reacts selectively with alcohols, aldehydes, ketones, and esters): Pre-prepared
solution of vanillin (1 g) and concentrated sulphuric acid (2 mL) diluted to a total volume of 100
mL using 95 % ethanol.
Determination of lovastatin yield: Accurately weigh a 10 mL pear-shaped flask (tare weight).

Transfer the fractions containing (pure) lovastatin into the flask and use dichloromethane to rinse
the tubes (pool with the rest). Remove the solvent using a rotary evaporator. Re-weigh the flask
afterwards.
𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑙𝑜𝑣𝑎𝑠𝑡𝑎𝑡𝑖𝑛 𝑠𝑎𝑚𝑝𝑙𝑒

% lovastatin = × 100%
𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑅𝑒𝑑 𝑌𝑒𝑎𝑠𝑡 𝑅𝑖𝑐𝑒 𝑠𝑎𝑚𝑝𝑙𝑒
61
Part C: NMR spectroscopic analysis and structure determination of sample with
isolated lovastatin
Lovastatin sample preparation: Dissolve your isolated lovastation sample in 800 L of deuterated
chloroform (CDCl3). Arrange a small amount of cotton wool as a filter in the neck of a Pasteur
pipette. Position the pipette in a 5-mm NMR tube and carefully filter your sample into the NMR
tube using a second Pasteur pipette for transfer. The sample must reach a depth of around 40-50 mm
in the tube. Mark the NMR sample with team number and group number, e.g., H1G13. This should
be written directly on the NMR tube no more than 4 cm from the top of the tube using a permanent
marker (ask for assistance if you are in doubt). Before placing the NMR tube in the rack for NMR
analysis, it should be used for part D described below.
Part D: Mass spectrometric analysis of sample with isolated lovastatin

Background information: Mass spectrometry (MS) allows the determination of the molecular mass
of a compound and may provide fragmentation patterns specific to that compound. High-resolution
mass spectrometry (HRMS) can provide so accurate masses that it is possible to calculate the
molecular formulae with minimum uncertainty. Unlike NMR spectroscopy, MS is a destructive
analytical method, but it requires much less material for analysis.
Sample preparation: Mark a HPLC vial with date, team number, and group number, and fill it with
500 L of methanol. Dip a capillary glass tube or Pasteur pipette in the NMR sample prepared
above (Part C), which by the capillary force will give 2-5 mm solvent in the capillary. Transfer this
amount to the 500 L methanol in the HPLC vial. Close the HPLC vial with a screw-cap and place
it in the rack for MS analysis. The sample will be subjected to HPLC-HRMS analysis.
Mass Spectrum: The expected signals of the mass spectrum of lovastatin are as follows:
• A peak corresponding to [M+H]+ and [M+Na]+ of lovastatin
62
Calculate the monoisotopic masses of these two ions and analyze the mass spectrum to see if they are
present. Note that you may observe other peaks than the above-mentioned.
The accuracy of the masses obtained in the high-resolution mass spectrum
Data analysis and report scheme

The LC-HRMS and NMR data of your sample will be available on Absalon approximately 24 hours
after the laboratory exercise – if no major instrumental breakdown occurs. All other data should be
collected (write down raw data like tare- and gross weights from weighing, record start and end
measurements from TLC, take photos, etc) by yourself during the exercise. A 1H NMR spectrum of
a reference sample of lovastatin and a paper from Journal of Organic Chemistry with 1H NMR
signals of common impurities and solvents (file named NMR chemical shifts) are available on
Absalon.

2. Experimental (special emphasis on changes to the experimental work compared to this exercise
manual)
3. Results and discussion highlighting the main findings from analysis of HRMS and NMR data of
the isolated material, including:
• Photos of TLC plate with fractions 15-30 monitored at 254 and 365 nm and your
reasoning for choosing fractions containing lovastatin
• Calculation of M (the mass error in ppm) for the experimental value of [M+H]+ relative
to the theoretical value and an evaluation of whether it is within the expected range.
• Discussion of identity and purity of your isolated material. Comparison of the 1H NMR
spectrum of your sample compared to the spectrum of a reference lovastatin sample
(available on Absalon) - and identification and discussion of residual solvent signals if
any (see file named 'NMR chemical shifts').
4. Short and clear conclusion about the work and the results.
The report should be uploaded to the assignment named 'Report exercise 5' with deadline
Friday October 14th.
63
We have…
 A photograph of the TLC plate at 254 and 365 nm after successful development.
 Noted down the fractions that were pooled for obtaining pure lovastatin.
 Prepared a sample in deuterated chloroform (CDCl3) for NMR analysis.
 Prepared a sample in methanol for LC-HRMS analysis.
Calculate the percentage yield of lovastatin relative to the total capsule content used:
Calculate the percentage yield relative to the theoretical content of lovastatin (as stated by the
manufacturer) in the capsules:
We have…
 Cleaned up our fume hood and washed dirty glassware
 Emptied waste containers in our fume hood and put a new waste bag in the container
Signature teacher
64
Exercise 6 – PTP1B inhibitors in Eremophila sp.
Time and place for mandatory exercise
This is a 'theoretical laboratory exercise', i.e. an exercise where you are provided a series of
experimental data acquired beforehand and where it is your task to process and analyse the data and
describe the results in a report. The theoretical exercise will take place in class rooms according to
your scheme:
Background
Eremophila is a genus of drought-tolerant plants endemic to Australia – especially the arid areas of
Western Australia. As part of a master project, a student investigated Eremophila denticulata for its
antihyperglycaemic effect. Thus, dilution series of a crude acetonitrile extract of leaves of Eremophila
denticulata were tested in -glucosidase, -amylase, and PTP1B (protein-tyrosine phosphatase 1B)
assays with the aim of obtaining sigmoidal inhibition curves for determination of IC 50 values. The
crude extract showed strong PTP1B inhibitory effect, and based on these results, it was decided that
the student should identify the compound(s) responsible for the observed PTP1B inhibitory activity.
Thus, it was decided that the following experiments should be performed:
• High-resolution PTP1B inhibition profiling of crude acetonitrile extract with the aim of
pinpointing analytes correlated with PTP1B inhibitory activity.
• HPLC-PDA-HRMS-SPE-NMR analysis to trap (isolate) and elucidate the structure of
analytes correlated with PTP1B inhibitory activity.
• Preparative-scale HPLC isolation of PTP1B inhibitory analytes and determination of IC 50
values for analytes with PTP1B inhibitory activity.
65
Preparation for theoretical exercise 6
Before the exercise, students should have prepared by reading Wubshet et al. J. Nat. Prod. 2016, 79,
1063-1072 which reports results from a study using similar methods. 21 Please make sure to have
downloaded and read the paper before the exercise.
Aim
The aim of this theoretical laboratory exercise is to familiarize the students with data processing and
interpretation of results from the combined use of analytical, chemical and pharmacological methods
for identification of bioactive natural products.
Experimental data available

The following experimental data is available:
• Data for biochromatogram: Retention times and UV data from the HPLC chromatogram as
well as retention times and bioactivity data from microfractionated data. These data can be found
in the Excel file named ‘Appendix 1 – Biochromatogram’
• Data for IC50 value: PTP1B slope data (rate of enzymatic hydrolysis) from a dilution series in a
96-well microplate with the compound at 31.4 min correlated with bioactivity in the
biochromatogram. These data can be found in the file named ‘Appendix 2 – IC50 value’
• HRMS data: A high-resolution mass spectrum of the compound at 31.4 min, which according
to the biochromatogram is correlated with bioactivity. This spectrum can be found in the file
named ‘Appendix 3 - HRMS spectrum’
• NMR data: 1H NMR, 13C NMR, DEPT135, COSY, HSQC, and HMBC spectra of the compound
at 31.4 min. These can be found in the file named ‘Appendix 4 - NMR data’
21
Wubshet, S.G.; Tahtah, Y.; Heskes, A.M.; Kongstad, K.T.; Pateraki, I.; Hamberger, B.; Møller, B.M.; Staerk, D.
Identification of PTP1B and -glucosidase inhibitory serrulatanes from Eremophila spp. by combined use of dual high-
resolution PTP1B and -glucosidase inhibition profiling and HPLC-HRMS-SPE-NMR. J. Nat. Prod. 2016, 79, 1063-
1072.
66
Tasks for exercise 6
Task 1 - Construction of a biochromatogram
In the file ‘Appendix 1 – Biochromatogram’ there are three sheets: i) CHROMATOGRAM with
retention times and corresponding absorbance values at 280 nm, ii) MICROFRACTIONATION
containing ‘Begin times’ and ‘End times’ for 88 microfractions in one 96-well microplates, and iii)
BIOACTIVITY containing percentage inhibition of these 88 microfractions. The first step in
constructing a biochromatogram is to make a HPLC chromatogram by making a X-Y scatterplot
with the data from the CHROMATOGRAM sheet. Subsequently you should take the average of the
‘Begin time‘ and ‘End time’ in MICROFRACTIONATION in a new column named ‘Average RT’
(to obtain one time-point for each fraction). Multiply the data in the BIOACTIVITY sheet with -1
and subtract 25 in a new column named ‘PTP1B inhibition trace’ to prepare for a PTP1B inhibition
profile with negative peaks and positioned below the X-axis. Now you should add the ‘Average RT’
and ‘PTP1B inhibition trace’ to the above X-Y scatterplot (the HPLC chromatogram), and the result
will be a biochromatogram, i.e. the PTP1B inhibition trace aligned with the HPLC chromatogram.
A short ‘how-to’ can be found in the ‘Guide to making Biochromatograms’ available on Absalon.
Task 2 - Determination of IC50 value
‘Appendix 2 – IC50 value’ contains slope data at 405 nm from triplicate measurements of a dilution
series in a PTP1B assay of material isolated from the peak at tR = 31.4 min in the biochromatogram
above. Read the description of the PTP1B inhibition assay in Experimental in the provided
manuscript by Wubshet et al., and use this knowledge to calculate percentage PTP1B inhibition for
the replicate measurements as well as to calculate the average percentage inhibition of the three
replicates. You now have a column with concentrations of the investigated compound and another
with its percentage inhibition. These two columns (or the concentrations and the replicate
measurements) should be used for calculation of the relative IC50 value - i.e. the concentration
found using the midpoint between the minimum and maximum y-values of a sigmoidal curve -
using a four-parameter fit:
max − 𝑚𝑖𝑛
𝑓(𝑥) = min +
𝑥 𝑠𝑙𝑜𝑝𝑒
1 + (𝐼𝐶 )
50
67
where min is the background, max − min is the y-range, x is the concentration and slope is the Hill
slope.
A series of commercial programs like GraphPad Prism, Grafit, etc are available for this, but here we
use the online IC50 calculator available at: https://www.aatbio.com/tools/ic50-calculator. You can
choose either to use the concentrations and the three replicates or the concentrations and the average
(note that the online IC50 calculator does not allow average ± SD). Type or paste your data into the
IC50 calculator and calculate the relative IC50 value. Make sure to take a screenshot of the IC50 curve
and the resulting four-parameter equation, where you can find the four parameters, including the
IC50 value.
Task 3 - Analysis of HRMS
‘Appendix 3 - MS data’ contains the high-resolution mass spectrum from a HPLC-PDA-HRMS-

SPE-NMR analysis of the material eluting with the peak at tR = 31.4 min in the biochromatogram
above. Identify the [M+H]+ ion and any adducts, and use the m/z-value of [M+H]+ to search
ChemCalc for possible molecular formulae. Note down M (the mass error in ppm) or calculate it
using the file 'MS calculations'). Next, search Reaxys for possible structures of natural origin with
your molecular formula, using the Form ‘Natural Products’ and select ‘Find any’. If in doubt,
refresh the dereplication process from class exercises 19 and 20.
Task 4 - Analysis of NMR data
‘Appendix 4 - NMR data’ contains 1D and 2D NMR data from a HPLC-PDA-HRMS-SPE-NMR

analysis of the material eluting with the peak at t R = 31.4 min in the biochromatogram constructed
above. Analyse the 1D and 2D NMR data (while still recalling your results from the Reaxys search)
and establish the structure of the unknown compound more or less following the process you have
practised in class exercises 13-18. However, note that signals in the 13C NMR spectrum are labelled
1-15, and you should use this numbering throughout your analysis and reporting of the results.
Furthermore, notice that you should use the below work flow and table to ease analysis and
reporting of results.
Work flow:
1) Fill in the 13C NMR values in column 3 of the below NMR table (Table 6.1, which can also be
found in the report scheme for exercise 6).
68
2) Use the HSQC spectrum to find one-bond C-H correlations, and fill in 1H NMR data (chemical
shift values, number of H's, coupling patterns and coupling constants) in the correct rows of
column 2 in Table 6.1.
3) Find 1H NMR signals without HSQC correlations and fill these data (chemical shift values,
number of H's, coupling patterns and coupling constants) into the last rows of column 2.
These protons do not have carbons directly connected, so therefore they appear in order of
increasing chemical shift values in the rows below no. 15.
4) Fill in the COSY correlations in column 4, i.e, write the 1H signals to which the protons in
column 2 correlate. Use the numbers from column 1.
5) Fill in the HMBC correlations in column 5, i.e, write the 13C signals to which the protons in
column 2 correlate. Use the numbers from column 1.
6) Based on the correlations from point 5), assign numbers in column 1 to those 1H NMR signals
without HSQC correlations from point 3). The easiest is to use e.g. “13-OH” for the OH group
on C-13, as it should be clear where in the structure it belongs.
7) Draw the chemical structure of the compound you by now have identified and assign numbers
(No. in the table below) to each 13C position of this structural drawing. It must be clear how
you assign each signal from the numbers in the structure.
Table 6.1. 1H and 13C NMR data of the peak at tR = 31.4 min and correlations found in 2D COSY and
HMBC spectra (numbering according to 13C NMR data).
Column 1 Column 2 Column 3 Column 4 Column 5

No.  1H (nH, multiplicity, JH,H (Hz))  13C COSY HMBC (H→C)
1 6.41 (1H, d, J = 2.1) 93.4 2 2, 3, 12, 14
2
3
4
5
6
7
8
9
10
11
12
13
69
14
15
-
-
-
-
-
The below 'Report scheme' should be filled out and uploaded to the assignment named 'Report
exercise 6' with deadline Friday October 21st
70
We have…
 Constructed a biochromatogram in Task 1
 Calculated percentage inhibition and determined the IC50 value in Task 2
 Saved a screenshot of the IC50 curve and IC50 regression results in Task 2
 Identified [M+H]+, adducts and losses in the HRMS spectrum in Task 3
 Calculated M in task 3 and compared it with the result from ChemCalc
 Begun the analysis of NMR spectra in Task 4
Signature teacher
71
Report scheme for exercise 6
FROM TASK 1:
Insert your biochromatogram as well as a short description of what information one can obtain from
the biochromatogram:
FROM TASK 2:
Show the output of your Excel file with calculated percentage inhibition and insert your IC50 curve
and regression results here:
Explain the meaning of IC50 in words:
FROM TASK 3:
Identify the m/z values of these ions:
[M+H]+ :
[M+NH4]+ :
[M+H−H2O]+ :
Can you identify other adducts or fragments?
72
What is M, the mass error in ppm? Is this result acceptable for identication by HRMS? What is
normally required?
FROM TASK 4:
Insert the correct chemical structure below! Which compound class does it belong to?
Insert results from your analysis of 1D and 2D NMR data in Task 4 in Table 6.1 below or directly in
the report scheme. Use the numbering from column 1 together with the structure. Furthermore, you
must prepare a figure showing HMBC correlations (arrows pointing from H to C) from the aromatic
as well as the hydroxyl protons to the carbon atoms they correlate with (if in doubt, see Figure 6 in
Wubshet et al.).
Table 6.1. 1H and 13C NMR data of the peak at tR = 31.4 min and correlations found in 2D COSY and
HMBC spectra (numbering according to 13C NMR data).
Column 1 Column 2 Column 3 Column 4 Column 5

No.  1H (nH, multiplicity, JH,H (Hz))  13C COSY HMBC (H→C)
1 6.41 (1H, d, J = 2.1) 93.4 2 2, 3, 12, 14
2
3
4
5
6
7
8
9
10
11
12
13
14
15
-
73
-
-
-
-
FROM TASK 1 THROUGH 4:

What are the three most important learning outcomes from this exercise?
1:
2:
3:
74

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UNIVERS ITY OF COPENHAGEN

FACULTY OF HEALTH AND MEDICAL SCIENCES

Drugs from Nature

Department of Drug Design and Pharmacology

Copenhagen – August 2022

Professor Dan Stærk

PRACTICAL INFORMATION ................................................................................................. 8

Laboratory safety ............................................................................................................................................................... 8

Groups and overview of exercises ..................................................................................................................................... 8

22 OBLIGATORISKE SIKKERHEDSSPØRGSMÅL .......................................................... 10

EXERCISE 1 – BOTANICAL EXCURSION AND HERBAL DRUGS ................................. 15

Schedule and meeting points ........................................................................................................................................... 15

Droge-øvelse på NaturMedicinsk Museum .................................................................................................................... 16

Spørgsmåls- og svarark til droge-øvelse på NaturMedicinsk Museum ...................................................................... 18

EXERCISE 2 – PURPLE CONEFLOWER HERB ............................................................... 20

Preparation for exercise 4................................................................................................................................................ 21

Corrections and comments to PURPLE CONEFLOWER HERB .............................................................................. 26

Data analysis and report writing .................................................................................................................................... 27

Post-lab checklist Exercise 2............................................................................................................................................ 28

Post-lab checklist Exercise 4.1.1 (week 37) .................................................................................................................... 29

Preparation for exercise 4................................................................................................................................................ 31

Data analysis and report scheme .................................................................................................................................... 36

Post-lab checklist Exercise 3............................................................................................................................................ 37

Post-lab checklist Exercise 4.1.2 (Week 38) ................................................................................................................... 38

Post-lab checklist Exercise 4.2.1 (Week 38) ................................................................................................................... 38

Report scheme for exercise 3 ........................................................................................................................................... 39

EXERCISE 4 – CHARACTERISATION OF FUNGAL CULTURES ................................... 41

Sources of filamentous fungi ........................................................................................................................................... 41

Secondary metabolite potential ....................................................................................................................................... 41

Identification of filamentous fungi.................................................................................................................................. 42

EXERCISE 4.1: ISOLATION OF ENDOPHYTIC FUNGI .................................................... 43

Exercise 4.1.1: Isolation of endophytic fungi – WEEK 37............................................................................................ 44

Materials for exercise 4.1.1: Isolation of endophytic fungi – WEEK 37 ..................................................................... 44

Experimental for exercise 4.1.1: Isolation of endophytic fungi – WEEK 37 .............................................................. 44

Exercise 4.1.2: Isolation of endophytic fungi – WEEK 38 and 39 ............................................................................... 45

EXERCISE 4.2: CHEMICAL INVESTIGATION OF AN ENDOPHYTIC FUNGUS ............ 46

Exercise 4.2.2: Chemical investigation of an endophytic fungus – WEEK 39............................................................ 49

EXERCISE 4.3: MORPHOLOGICAL INVESTIGATION OF FILAMENTOUS FUNGI ....... 51

Data analysis and report writing for exercises 4.1-4.3.................................................................................................. 54

Post-lab checklist Exercise 4.1.2, 4.2.2 and 4.3 (Week 39)............................................................................................ 55

EXERCISE 5 – ISOLATION OF LOVASTATIN FROM RED YEAST RICE ...................... 56

Part A: Preparation of crude red yeast rice extract ..................................................................................................... 59

Part B: Isolation of lovastatin by flash chromatography ............................................................................................. 59

Data analysis and report scheme .................................................................................................................................... 63

Post-lab checklist Exercise 5............................................................................................................................................ 64

EXERCISE 6 – PTP1B INHIBITORS IN EREMOPHILA SP. .............................................. 65

Preparation for theoretical exercise 6 ............................................................................................................................ 66

Experimental data available ............................................................................................................................................ 66

Tasks for exercise 6 .......................................................................................................................................................... 67

Post-lab checklist Exercise 6............................................................................................................................................ 71

Report scheme for exercise 6 ........................................................................................................................................... 72

Groups and overview of exercises

NOTE: Laboratory exercises start at 12:30 and ends at 16:30

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