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MOI UNIVERSITY

ISO 9001:2015 Certified Institution

INDUSTRIAL ATTACHMENT REPORT

By

Biochemistry

Reg No: BIO/18/17

An Industrial Attachment Report submitted in partial fulfilment of the requirement

for the award of Bachelor of Science Degree in Biochemistry

SCHOOL OF SCIENCE AND AEROSPACE STUDIES

DEPARTMENT OF CHEMISTRY&BIOCHEMISTRY

April 2020
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ATTACHMENT DETAILS

Course Code: BIO 320

Course Title: INDUSTRIAL ATTACHMENT

Institution Attached: KENYA MEDICAL RESEARCH INSTITUTE

Attachment Start Date: 07/01/2020

Attachment End Date: 31/03/2020

Student Signature: -------------------------------------------- Date:

------------------------------------------
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REPORT SUMMARY

The industrial attachment was a mind thrilling experience with lots of learning opportunities. As

a biochemist, having undertaken an attachment at KEMRI I was exposed to new skills and got

the hands-on skills on theoretical concepts we had learned earlier.

The report shares experiences in the field of attachment discussing in details what was taught in

the laboratories. KEMRI being a medical research institute, we had the opportunity to carry

research on some of the epidemics that have continued to be a challenge in Kenya amongst them

being malaria. Amongst other fields of interest was the phytochemical screening of various plant

extracts in the determination of their potency in management of ailments such as malaria and

cancer.

However, not all the details of the attachment experience have been included in this report as

most of it has been recorded in the student’s attachment logbook. Amongst other important

details of the attachment experience is the opportunity to work on nano formulations. As

explained in the report, nano particles are particles of very small size and are used in medicine to

aid in drug delivery thus increasing bioavailability, target specificity and reduced toxicity.

Important figures, diagrams and charts have been added to aid and ease in interpretation of data

and increased understanding of procedures and processes.

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LIST OF ACRONYMS AND ABBREVIATIONS

KEMRI- Kenya Medical Research Institute

CTMDR- Centre for Traditional and Drugs Research

CMR- Centre for Microbiology Research

CVR- Centre for Virus Research

DMSO- Dimethyl Sulfoxide

MTT- Tetrazolium dye.

CDC- Centre for Disease Control

CSC- Centre Scientific Committee

THP-Traditional Health Practitioners

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LIST OF FIGURES

Figure 1: Organizational structure for KEMRI CTMDR ………………………......Pg.10

Figure 2: Hela cells as seen in an inverted light microscope …………………….... Pg.13

Figure 3: cryopreservation and cell passaging process…………………. Pg.14

Figure 4: Thin layer chromatography for Bredelia micaranthus …………………………Pg.15

Figure 5: extracts stored in vials…………………………………………. Pg.16

Figure 6: Entry of daily records illustrated………………Pg.19

Figure 7: Sodom apple images…………………………….……. Pg.20

Figure 8: Schistosoma mansonii life cycle illustrated……………………………………. Pg.23

Figure 9: images of the cell cycle of Schistosoma spp………………………………Pg.24

Figure 10: schistosomiasis…………………………………………….... Pg.24

Figure 11: A hemocytometer grid ………………………………………. Pg.25

Figure 12: A microtiter plate after an MTT assay……………………. Pg.26

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TABLE OF CONTENTS

Table of Contents

REPORT SUMMARY.....................................................................................................3

LIST OF ACRONYMS AND ABBREVIATIONS............................................................4

LIST OF FIGURES ...............................................................................................5

TABLE OF CONTENTS........................................................................................6

INTRODUCTION AND OBJECTIVES .................................................................................7

INFORMATION ABOUT THE INSTITUTION OF ATTACHMENT.......................................9

ACTIVITIES AND EXPERIENCES ...............................................................................11

INTERACTION WITH INSTITUTION’S STAFF..........................................................17

CHALLENGES ENCOUNTERED......................................................................................17

ORGANIZATION OF DAILY RECORDS ......................................................................18

ENTRY OF WEEKLY ACTIVITIES ...................................................................................23

RECOMMENDATIONS...........................................................................................29

REFERENCES .................................................................................................................30

APPENDICES .....................................................................................................................31
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INTRODUCTION AND OBJECTIVES

PURPOSE OF THE ATTACHMENT

The industrial attachment is an important program to the students and especially to science

students who need the hands-on skills on various scientific machineries. The program is aimed at

instilling the students with skills and experience and more learning so as they have an impact in

the science world.

As a biochemist, being attached at KEMRI helped me apply the theoretical teachings from

school and also get the hands-on skills on various biochemistry techniques. These included

development of medicines in treatment of ailments such as cancer and malaria, carrying out of in

vivo and in vitro tests, cell passaging, cell density count and phytochemical screening of herbal

plants.

OBJECTIVES OF THE ATTACHMENT COURSE

The Centre for Traditional Medicine and Drug Research (CTMDR), is divided into different

units each tasked with responsibilities that together purpose to better the standards of drugs in the

health sector. By the end of the attachment program I was able to achieve the following

objectives:

a) Carrying out of literature review on certain medicinal herbs.

b) Extraction of compounds from plants using various extraction methods.

c) Phytochemical screening to test the presence of alkaloids, terpenoids, saponins and

phenolic compounds.

d) Carrying out in vivo and in vitro tests of medicinal herbs against malaria.

e) Cell passaging and cryopreservation of cancer cells, i.e. colon cancer cells (CT 26 CRI

2638) and Hela cells.

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f) Cell density determination using the trypan blue exclusion method.

g) Preparation of empty and loaded nanoparticles using praziquantel drug. The praziquantel

drug is used in the management of schistosomiasis.

h) Preparation of solutions of drug materials using solvents. The most commonly used

solvent was DMSO.

i) Carrying out of cytotoxicity tests using MTT assay and recording the elisa readings.

j) Carrying out herbal medicine formulation studies.

k) Determination of quality of pharmaceutical products by identifying the molecular

structure of the drug’s pure compound.

Getting an opportunity to achieve these objectives prompted me into wanting to work at the

institution in the near future. Furthermore, during the last weeks of our attachment program, we

went through a COVID-19 sensitization training which gave me an interest in studying virology.
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INFORMATION ABOUT THE INSTITUTION ATTACHED

KEMRI stands for Kenya Medical Research Institute. It is a research institute that has been doing

research on human health for over forty years. The institutions motto, (in search for better health)

drives the scientists at the institution into working harder in achieving its greater vision which is

to improve the quality of health through research, innovation and capacity building.

The institution has various centers located in different parts of the country. At Nairobi, the

following centers are located there;

a) Centre for Traditional Medicine and Drug Research (CTMDR)

b) Centre for Microbiology Research (CMR)

c) Centre for Virology Research (CVR)

d) Centre for Disease Control (CDC)

e) Centre for Clinical Research (CCR) among others.

At CTMDR, where I was attached, there are five units namely;

1. Chemical sciences unit.

2. Biological sciences unit.

3. Pharmaceutical sciences.

4. Botanical sciences.

5. Administration.
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ORGANAZATION STRUCTURE

Fig.1. organizational chart for CTMDR.

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ACTIVITIES & EXPERIENCES

At the Centre for traditional medicine and drug research, we were taken through an orientation

program where the various units of the center were introduced amongst other details including

arrival time and the activities, we would be involved in. the following is a detailed explanation of

the activities of each unit:

A. Botanical sciences unit

i. The unit is the link between traditional health practitioners (THPs) and the

center

ii. Carries out literature reviews of medicinal herbs and documents the medicinal

herbs they have done research on.

iii. Involves with the collection of herbs from the field, grinding, conservation

and storage of the ground material.

B. Chemical sciences unit

i. Phytochemical screening of extracted material through procedures such as

thin layer chromatography and column chromatography.

C. Biological science unit

i. The unit is in charge of the animal house where they breed mice used in in

vivo tests. At the animal house, they have the swiss albino mice, balb mice

and hamsters.

ii. Carries out in vitro tests, cell passaging and cryopreservation of cells.

iii. Carries out nano formulation studies. This involves development of nano

particles. Nano particles are particles of very small size. In medicine and
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pharmaceutical industries, nano particles are used to aid in drug delivery. nano

formulation of drugs improves pharmacokinetics, bioavailability, solubility

and reduced drug toxicity.

D. Pharmaceutical sciences

i. Carries out herbal medicine formulation studies.

ii. Pilot production of herbal medicines.

iii. Determination of quality of pharmaceutical products.

iv. Carrying out disintegration studies.

v. Carrying out dissolution studies.

vi. The unit also performs formulation studies, such formulations include;

capsulation, making of skin care products e.g. soaps, creams and ointments
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Fig.2.

HELA cells as seen under inverted light microscope.

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Fig.3.

Cell passaging and cryopreservation procedure.

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Fig.4.

Thin layer chromatography done on a TLC plate.

The plate is prepared using silica gel. Silica gel and distilled water are mixed in the ratio 1:2, the

gel is then spread on the plates and air dried and then activated by heating in an oven at 100-

110°C.

The figure shows separation of a concentrated extract of Bredilia micaranthus.

The distinct fractions are then collected and a solvent extract of DCM/methanol added to

dissolve silica gel. The contents are filtered using a filter paper and the filtrate stored in vials as

shown in the figure below.

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Fig.5.

Extracts stored in vials.

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INTERACTION WITH INSTITUTION’S STAFF

When we were ushered in at the Centre, we were introduced to the research scientists at the

Centre, lab technicians, interns, students doing research at the Centre and also support staff. We

were treated as institutions staff and got a chance to even attend the Centre’s meetings including

the Centre scientific committee meeting. The CSC is a scientific review board within the Centre

whose role is to review research proposals and manuscripts for publication on scientific journal

sites e.g. science direct, PubMed among others.

The institutions staff were supportive and ready to help us learn. The technicians took us through

the lab sessions and explained all procedures in details.

CHALLENGES ENCOUNTERED

a. Inadequate resources

The Centre lacked some of the instruments and machinery used in research. For instance,

we had to move to another Centre to concentrate samples after extraction since the Centre

lacked a Rota vapor.

b. Some biochemistry techniques were introduced while on attachment. These techniques

were only taught in theory at school.

c. We performed some of the lab procedures in incomplete due to expensiveness of the

materials and reagents required.

d. The covid-19 crisis was also a challenge as it cut short our learning experiences before

our attachment completion date.

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ORGANIZATION OF DAILY RECORDS

All the daily activities were recorded in the log book and a description of the work done made.

Daily records were entered in the evenings upon completion of the day’s activity. On the days

that we had no lab sessions, the day’s activity was recorded according to the previous days’

homework or on the academic research done on that particular day.

Meeting records were also indicated stating the business of the day and the agenda of the specific

meetings. On days that we weren’t present, say on weekends, the statement on the days record

would be N/A implying that no work as done on that day.

The table below shows an example of how daily records were done.

WEEK 2 PROGRESS ACTIVITY/DESCDRIPTION

CHART. WEEK ENDING OF WORK DONE

24/01/2020
MONDAY Under the botanical science unit,

a detailed procedure on

ethnobotanical survey was

discussed. These included

identification of medicinal

plants to documentation of the

same.
TUESDAY Conducting literature review on

Sodom apple. The scientific

name for Sodom apple is

solanum incanum.
THURSDAY A detailed description of

standard operating procedures in

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the botanical science unit was

discussed. These included an

SOP on sample collection

procedure, grinding and storage

of the collected sample.

FRIDAY Visited the botanical storage

unit where we learnt on

documentation of the grounded

powder. The powder is stored in

bags and sealed. The bag is then

labelled indicating the name of

the plant and the weight of the

powder.
Fig.6...Entry of daily records.

The literature review conducted on Solanum incanum was a detailed description ranging from

the distribution of the plant to its pharmaceutical uses.

LITERATURE REVIEW OF SODOM APPLE.

BOTANICAL NAME: Solanum incanum

FAMILY: Solanaceae

COMMON NAMES: bitter apple, Sodom apple.

LOCAL NAMES: Mutongu (Meru and kikuyu), Etulerut (Teso), Ochok (Luo), Labotwa
(Marakwet), Mtunguza Koma (Digo), Mutungu (Kamba), Maduranzura (Luhya), Mtunguja
(Swahili). (kokwaro)

BOTANICAL DESCRIPTION
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Solanum incanum, is a perennial, wild shrub like herb that belongs to Solanaceae family which
grows in many regions of Africa, Middle East and Far East Asia.

It is an erect or spreading perennial shrub with leaves and stem occasionally having small
prickles. The fruits are small berries of 2-3 cm in diameter and yellowish orange or brown in
color when ripe (Matu 2008)

PHOTO

Fig.7.sodom apple

DISTRIBUTION
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olanum incanum is abundant and

common as a
solanum incanum is abundant and common as a weed, around houses, in overgrazed grassland
and on roadsides. It is also found at forest edges and in bushland and grassland from sea level up
to 2500 m altitude. It is widely found in eastern western and central parts of Kenya (Mwonjoria
J.K).

olanum incanum is abundant and

common as a
TRADITIONAL MEDICINAL USES

1. The herb is used as a folklore remedy for sore throat, angina, stomach-ache, colic,
headache (Kokwaro 1993).
2. The roots of the plant are used for abdominal pains, dyspepsia, fever, stomach ache, and
indigestion (Kokwaro 1993).
3. The roots can also be used for toothache by scrubbing the affected tooth with pieces of
the root (Kokwaro 1993).
4. Young leaves are chewed and rubbed hard into a recent snake bite (Kokwaro 1993).
5. An infusion of the leaf is applied to the ear as remedy for earache (Kokwaro 1993).
6. The contents of the fruits are used for fresh cut wounds (Kokwaro 1993).
7. Used for chest pains, ringworm and syphilis (Kokwaro 1993).
8. Treatment of skin diseases (Kokwaro 1993).

PHARMACOLOGICAL ACTIVITY

In addition to the several important traditional medicinal applications of solanum incanum, the
plant is a rich source of important cytotoxic glycoalkaloids such as solarmargine and solasonine.
The herbal extract posses’ antinociceptive, antipyretic, ant spasmolytic, orexic, anorexic,
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hypoglycemic, antimicrobial, anti-schistosomal, anti-fungal, and anti-cancer activity (Mwonjoria

J.K et.al)

Solasonine and solamargine inhibit plant growth (kokwaro).

CHEMICAL CONSTITUENTS

Some of the chemical components contained within solanum incanum include; incanumine,
solasodine, carpesterol, adenosine, glycopyranoside, khasianine, Solasonine, solamargine among
others (Eltayeb et al. 1997).

TOXICITY

The highest concentration of the alkaloid is found in the smallest leaves of S. incanum while the
roots and stem were initially shown to have similar concentration of the alkaloid (Eltayeb et al.
1997). The fruits contain dimethyl nitrosamine, a potent carcinogen that may be associated with
high incidence of esophageal cancer in areas of Africa where the fruit sap is used to curdle milk.
Similarly, extracts of the fruits has been found to cause skin cancer in animals.
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ENTRY OF WEEKLY ACTIVITIES

Weekly activities were recorded being a compilation of the daily activities of that specific week.

This included a weekly report stating what had been done on that specific week. In the report, a

detailed description of the daily activities was made. Any sketches or figures that explained

further on the daily records were drawn and well labelled.

For instance, on week seven of our attachment we visited the animal house and learnt on the life

cycle of Schistosoma spp. Therefore, I deemed it important to include a figure indicating the life

cycle of Schistosoma spp, right from when the eggs are shed from an infected human to the

migration of the cercariae to portal blood in liver and maturation into adults.
IG
M
O
P
S
T
H
N
A
C
U
L
Y
R
E
D

Fig.8. life cycle of Schistosoma spp.

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Fig.9. images of the cell cycle of Schistosoma spp.

Fig.10. schistosomiasis.

CELL DENSITY DETERMINATION

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During the same week, we did cell density determination using the trypan blue exclusion

method. During the process a hemocytometer is used to calculate the number of viable cells per

grid. For instance, a definite volume of cell suspension was taken i.e. 50µl and 100µl of trypan

blue added. Trypan blue is added to stain the cells as the dead cells pick up the stain. A

hemocytometer is then used to calculate the number of viable cells per grid.

CALCULATIONS

A. Dilution factor (df)= x +50÷ 50

=150 ÷50=3

B. Average number of cells;

=80÷8=10
Cell density per ml
= A × DF ×10 4
WHERE; A-average number of cells
Df-dilution factor
=10 ×3 ×10 4=3.0 ×105 cells /ml

Fig.11. A hemocytometer Grid.

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A MTT assay is run to determine the number of viable cells. MTT, a yellow tetrazole is reduced

to purple formazan in living cells. The living cells have enzymes that are capable of reducing the

tetrazolium dye to its insoluble formazan which is purple in color.

Fig.12. A microtiter plate after an MTT assay. Increasing amounts of cells resulted in increased purple coloring.

PHYTOCHEMICAL SCREENING

Screening for phytochemicals helps in learning the phytochemistry of medicinal plants which is

vital in understanding the therapeutic uses of the plant. There are various phytochemicals

screened in plants which include; alkaloids, terpenoids, phenolics, saponins etc.

SCREENING FOR ALKALOIDS

This is done using the dragendoff solution. After running TLC, the plates are sprayed using the

solution. Orange brown spots on the yellow background indicated presence of alkaloids.

TEST FOR PHENOLICS

This is done by spraying equal parts of 1% ferric chloride and 1% potassium ferricyanide on the

plates. A blue color gave an indication of phenolics presence.

TEST FOR SAPONINS

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A small amount of the extract was put into a test tube and then distilled water added. The

contents are then shaken vigorously for about a minute and observations made. When a ring of

foam was observed that lasted for long, it indicated presence of saponins.

SCREENING FOR TERPENOIDS

Detection of terpenoids is done using 1% vanillin in concentrated H 2 SO4 . After running a TLC,

the plates are sprayed using vanillin. Any color change gave an indication of terpenoids

presence.

NANO FORMULATION

On week five of our attachment, we interacted with a research scientist at the centre who took us

through the nano technology. This entails formulation of nano particles which are mostly used in

medicine to aid drug delivery. Nano particles are particles of very small size. Nano particles are

used in medicine since nano formulated drugs are advantageous in;

a. Maintaining drug therapeutic concentrations.

b. They are more target specific.

c. Leads to improved bioavailability, solubility and pharmacokinetics.

d. Reduces the toxicity of the drugs.

Nano particles are prepared in the laboratory using a double emulsion solvent evaporation

technique. The technique involves two phases; organic and aqueous phase. the organic phase is

prepared by dissolving 50mg of stearic acid in 10ml ethyl acetate. The contents are stirred using

a magnetic stirrer. The aqueous phase is then prepared using pre-prepared 10ml of polyvinyl

alcohol (PVA), 5ml of 0.2% chitosan and 5ml of 5% lactose. PVA was prepared by dissolving in

water for three hours, while chitosan was left overnight in water to dissolve.
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The aqueous phase is stirred using a magnetic stirrer and then added to the organic phase. they

are then homogenized for 3 minutes at 6000rpm. A second aqueous phase is prepared using 10ml

PVA, 5ml chitosan and 5ml lactose and added to the first mixture. The resulting water-in-oil-in-

water emulsion is further emulsified for three minutes by homogenizing at 10,000rpm. When

homogenizing, the beaker is placed in a bowl full of ice due to high temperatures. The aqueous

phase volume is higher so as to ensure it covers the organic phase.

The empty nano particles obtained were characterized for particle size and polydiversity index

(PDI). Each sample was measured in triplicate.

When loading the praziquantel, the drug is introduced in the organic phase since it is insoluble in

water. However, ideally it should be introduced in the aqueous phase.

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RECOMMENDATIONS

a) the school should include more field trips to research centers as they expose the students

into getting the hands-on skills and experience.

b) The school should build a research Centre which is well equipped. This will not only help

the students but also create employment to the thousands of research scientist in the

country.

c) The department of chemistry and biochemistry should partner with CTMDR in doing

research on traditional medicines in management of ailments such as malaria.

d) The school in partnership with science student’s association, should organize research

conferences and invite institutions like KEMRI, KARLO, KEPHIS, IRDI among others.

e) The government of Kenya should build more research centers to better the state of health

by promoting research on human health.

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REFERENCES

1. Matu E.N (2008), Solanum incanum L, PROTA (Plant Resources of Tropical Africa),
Wageningen Netherlands, Protabase.
2. Kokwaro J O (1993), Medicinal plants of East Africa, 2nd Edition East Africa Literature
Bureau, Nairobi.
3. Eltayeb EA, Al-Ansari, Roderick JG (1997), Changes in steroidal alkaloid Solasodine
during development of Solanum nigrum & Solanum incanum, Phytochemistry 46(3)
489– 494
4. Mwonjoria, JK, Kariuki H and Waweru FN (2011), the antinociceptive antipyretic effects
of Solanum incanum (Lin.) in animal models, Intern. J. Phyto pharm.
5. Mwonjoria, JK, (2014) Ethnomedicinal, phytochemical and pharmacological aspects of
solanum incanum, International Journal of Pharmacology and Toxicology, 2 (2) (2014)
17-20.
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APPENDIX 1: KEMRI- CTMDR Standard operating procedure for collecting of plant

materials the botanical science unit

1. Conduct prior literature search from collection at the East African herbarium on sites

where the plants of interest have been collected.

2. Assembling the materials required in the field e.g. panga.

3. Once in the field, apply sustainable harvesting techniques. E.g. avoid ring barking, collect

minimal side roots etc.

4. Collect a voucher specimen and press it in a plant press. Give it a reference number of

choice.

5. Pack the collected materials into a sisal gunny bag for transportation to the lab within

three days.

6. Once in the lab, spread the plant materials on a clean sisal gunny bag in a airy room and

allow it to dry for 14 days. Label the gunny with the name of the plant.

7. Once dry, take the plant materials for grinding.

8. After grinding into powder, weigh the grounded material and store in a packaging manilla

paper. The manilla paper should indicate the name of the owner, name of the plant

species, part of the plant, weight of the plant material, site and date of collection.
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APPENDIX 2: KEMRI- CTMDR Laboratory procedure for preparation of drag doff

solution.

a. Dissolve 0.85g of bismuth subnitrate in a solution of 10ml acetic acid and 40ml water.

b. Dissolve 8g of potassium iodide (KI) in 20ml of water.

c. Take equal parts of (a) and (b). mix equal parts of the stock solution (a) and (b) 1ml each

and add 2ml acetic acid and 10ml water.