Drug Discovery and Development Drug Discovery
Prospects and Challenges
This book is written to provide a brief overview concerning the latest drug and Development
discovery and its development based on the research studies that have been
carried out until the present. The idea of preparing this book was inspired by
the Holy Qur’an and the hadith, which guides us in all aspects of life, Prospects and
including disease management and treatment. This was supported by the
traditional use of natural medicine by our ancestors. From the scientific
point of view, we translate all those knowledge and information to be Drug Discovery and Development Prospects and Challenges Challenges
evidence-based modern medicines. We practised the common process of
drug discovery development by identifying the potential molecule,
followed by in vitro and in vivo experiments. The formulation is a crucial
process to produce an efficient and effective drug delivery. Not to forget the
future vision that “things would not give any meaning if it is not giving
benefit to the people”. Commercialisation is one of the approaches to
promote the product across the market and community. The book ends with
the current issue of the COVID-19 global pandemic with regards to the
treatment, natural medicine, and vaccination.
MUHAMMAD TAHER BAKHTIAR completed his Bachelor of
Pharmacy from University of Andalas in 1997. He obtained Master of
Science in organic chemistry in 2000 and Ph.D in bioprocess in 2005 from
the University Teknologi Malaysia. Since April 2006, he joined the
International Islamic University Malaysia (IIUM). He established animal
cell culture laboratory at the Kulliyyah of Pharmacy. He gained experience
in administrative positions such as CPD Coordinator for Kuantan Campus, MUHAMMAD TAHER BAKHTIAR
Deputy Dean Postgraduate Students, Head of Research and later as Head
Department of Pharmaceutical Technology. His research interest is in drug
discovery involving extraction, isolation, characterization and bioactivity MUHAMMAD TAHER BAKHTIAR
study of natural products from plants.
e ISBN 978-967-491-154-6
978967491154 6
PROF. DR. MUHAMMAD TAHER BAKHTIAR
PROF. DR. MUHAMMAD TAHER
2
(Ph.D., MSc., BPharm., Apt.)
Kulliyyah of Pharmacy, IIUM
[email protected]
Gombak • 2021
First Print, 2021
© IIUM Press, IIUM
IIUM Press is a member of the Majlis Penerbitan Ilmiah Malaysia - MAPIM
(Malaysian Scholarly Publishing Council)
Persatuan Penerbit Buku Malaysia - MABOPA
(Malaysian Book Publishers Association)
Membership No. - 201905
All rights reserved. No part of this publication may be reproduced, stored in
a retrieval system, or transmitted, in any form or by any means, electronic,
mechanical, photocopying, recording, or otherwise, without any prior
written permission of the publisher.
Perpustakaan Negara Malaysia Cataloguing-in-Publication Data
Muhammad Taher Bakhtiar
Drug Discovery and Development: Prospects and Challenges /
Muhammad Taher Bakhtiar
ISBN 978-967-491-153-9
E-ISBN 978-967-491-154-6
1. Pharmaceutical industry
2. Biotechnology industries
3. Pharmacy—Research
4. Pharmaceutical biotechnology
5. Drugs—Research
6. Drug development
7. COVID-19 (Disease)—Treatment
338.476151
HD9665.5
Published in Malaysia by
IIUM Press
International Islamic University Malaysia
P.O. Box 10, 50728 Kuala Lumpur
Contents
Summary vii
Introduction 1
Natural Medicines According to Islamic and Old Malay
Scriptures 3
Progress in Pharmaceutical Development 5
Source of Drugs 7
Journey in Research: A Personal Experience 9
Isolation and Characterisation Studies 9
In Vitro and In Vivo Studies 13
Anti-cancer/Cytotoxicity 14
Anti-hyperglycemic 15
Anti-obesity 19
Wound Healing 20
Drug Development Process 21
Importance of Animal Study 22
Drug Formulation 23
The Miracle Plant, Centella asiatica 27
Introduction of C. asiatica 27
Wound Healing Process 29
Hydrogel 30
Development of Pegaga Research: From Laboratory to Serum X 31
Hydrogel Formulation using C asiatica 31
Pegaga Extraction 32
Optimisation of Fractionation 32
HPLC Analysis 34
Upscale Production of Pegaga Hydrogel 35
vii / Contents
Pre-commercialisation of Pegaga Serum 36
Output from Pegaga Research 37
The COVID-19 Pandemic 37
Conclusion 40
Acknowledgements 41
Bibliography 45
Curriculum Vitae 55
Summary
Drug discovery and development is a vital process before a new drug
can be introduced for clinical use. Undeniably, the process to produce a
single drug that is safe for the human body and effectively treat specific
disease without any side effects is lengthy and very costly. It starts from
the curiosity over an emerging disease or the absence of effective medi-
cation and is translated into a scientific and clinical study through various
research approaches, such as from jungle-to-bench, from culture-to-vial,
and from waste-to-wealth.
Since ancient times, humans have produced drugs or medicine from
raw plant materials and their extracts to treat various illnesses. Today, the
paradigm has shifted from test and trial to a scientific evidence-based ap-
proach through experiments and clinical trials. High throughput methods
are also crucial tools to speed up the process of drug discovery. Various
drugs have been introduced via stringent guidelines and regulations. With
the emergence of new diseases, the development of new side effects, and
the latest Coronavirus Disease 2019 (COVID-19) outbreak, these expe-
riences and scenarios have taught us how to address the global impact
of disease through collaboration and synergistic strategies. As part of the
intellectual community, our collective knowledge would be meaningful
to contribute to the discovery and development of effective drugs.
The main obstacle in drug discovery and development nowadays is
related to biological/pharmacological activities and delivery methods.
Given this, nanotechnology has provided an impactful approach to drug
and vaccine delivery. Certain drugs that possess unwanted side effects can
be improved through nanotechnology to reduce their side effect, while
their effective constituent can be delivered to the specific site in a very
effective manner. Furthermore, the development of nanocarriers, such as
nanovesicles, nanoemulsions, and polymers, has become a global interest
due to its ability to increase, protect, and encapsulate drugs, ultimately
increasing the efficacy of drug delivery.
ix / Summary
Therefore, drug discovery and development require a continuous
supply of innovative ideas, effective strategies, and strong synergy to
improve the quality of modern medication and provide a reliable treat-
ment against new diseases. The collaboration between academia and
industry would not only strengthen the efforts to develop and discover
new drugs but also avoids a gap in the development process, which may
lead translational research to a valley of death.
Assalamu’alaikum warahmatullahi wabarakatuh and salam sejahtera,
Honorable Datuk Dr. Mohd Daud Bakar, President of the International
Islamic University Malaysia,
Honorable Prof. Emeritus Tan Sri Dato’ Dzulkifli Abdul Razak, Rector
of the International Islamic University Malaysia,
Honorable Deputy Rectors, the International Islamic University
Malaysia,
Respected University Management Committee, Deans, Directors,
Senior Officers, Professors, Lecturers,
Brothers and sisters,
Ladies and Gentlemen,
Introduction
The human being is the best creation by Allah subhanahu wa ta’ala
(Glory to Him, the Exalted). We are asked to seek knowledge with the
‘aqal (intellect/reasoning) that Allah has given to us. There are many
verses in the Holy Qur’an that encourage us to explore the secret of
nature. As mentioned in Surah Al-Baqarah: 164, which reads:
“Indeed, in the creation of the heavens and the earth; the
alternation of the day and the night; the ships that sail the sea
for the benefit of humanity; the rain sent down by Allah from
the skies, reviving the earth after its death; the scattering of all
kinds of creatures throughout; the shifting of the winds; and the
clouds drifting between the heavens and the earth—[in all of
this] are sure signs for people of understanding”. (164)
The phenomenon on the earth should be studied with faith, as it has
been done by the Islamic scholar, Jabir ibn Hayyan, famously known as
the father of chemistry and one of the founders of modern pharmacy. He
had written 300 books on philosophy, 1300 books on mechanical devices,
and hundreds of books on alchemy. Jabir is credited for his experimental
methodology in chemistry and the introduction of chemical processes in
2 / Drug Discovery and Development: Prospects and Challenges
modern chemistry (Amr & Tbakhi, 2007). As quoted in Surah Ali Imran:
190–191, Allah said:
“Indeed, in the creation of the heavens and the earth and the
alternation of the night and the day are signs for those of
understanding”. (190)
“Who remember Allah while standing or sitting or [lying] on
their sides and give thought to the creation of the heavens and
the earth, [saying], “Our Lord, You did not create this aimlessly;
Exalted are You [above such a thing]; then protect us from the
punishment of the Fire”. (191)
In these verses, the ulul al-bab (people of intellect) are the people
who performed tadzakkur (remembering Allah), and tafakkur (thinking
of Allah’s creation). In other words, thinking and research are the souls
of knowledge. Islam has a clear guideline and framework to process
ijtihad (independent reasoning) in solving human interest through
Maqasid al-Shariah (the objectives of sharia), which includes ijtihad
in protecting life. From a pharmaceutical point of view, this ijtihad
encourages the effort to research on medicine and health, which can be
performed through drug discovery and development. With regards to
the COVID-19 pandemic, as a Muslim, we shall refer to the sayings of
Prophet Muhammad (Peace Be Upon Him):
“There is no disease that Allah has created, except that Allah
also has created its cure.” (Sahih Bukhari)
The COVID-19 has affected every single human life and activity. It
has also affected the teaching and learning process at the higher education
level. In line with that, IIUM has come up with the Sejahtera Academic
Framework, which emphasises the strategic plan to be resilient during
and post-pandemic. Therefore, our duty as academics is to play a part
in the war against COVID-19 by finding an effective cure through
research and development. With no guarantee that a potential pandemic
would occur in the foreseeable future, we should prepare ourselves with
science and an evidence-based approach to seeking possible solutions
and effective medications, either through natural or traditional sources
or the development of high-tech drugs.
Drug Discovery and Development: Prospects and Challenges / 3
Brothers and sisters,
Ladies and Gentlemen,
Natural Medicines According to Islamic
and Old Malay Scriptures
The Islamic scriptures consisting of the Holy Qur’an and the hadith
describe a number of natural remedies, such as Allium cepa L. (onion),
A. sativum L. (garlic), Phoenix dactylifera L. (date), Lagenaria siceraria
Standl. (pumpkin), Ficus carica L. (fig), Olea europea L. (olive), Lens
culinaris Medic (adas), Hordeum vulgare L. (barlie), Punica granatum
L. (pomegranate), Salvadora persica L. (siwak/miswak), Vitis vinifera L.
(grape), and Zingiber officinale Roscoe (ginger) (El-Seedi et al., 2019).
All the aforementioned natural medications have gained the interest of
the global research community due to their healing efficacy. The number
of scientific papers related to these natural remedies from the Scopus
community due to their healing efficacy. The number of scientific papers related to these natural
database is shown in Figure 1.
remedies from the Scopus database is shown in Figure 1.
Number of articles
25,000
20,000
15,000
10,000
5,000
0
Hordeum vulgare
Allium sativum
Allium cepa Phoenix dactylifera Ficus carica Olea europea Lens culinaris Punica granatum Vitis vinifera
Zingiber officinale
Salvadora persica
Lagenaria siceraria
Figure 1: Publications related to natural medications that were
Figure 1: Publications related to natural medications that were mentioned in the Holy Qur’an and the
mentioned in the Holy Qur’an and the hadith
hadith
In the Malay Archipelago region (Malaysia, South Thailand,
In the Malay Archipelago region (Malaysia, South Thailand, Singapore, Brunei Darussalam,
Singapore, Brunei Darussalam, and Indonesia), medicinal plants have
and Indonesia), medicinal plants have been utilised in the form of jamu to heal various ailments. Jamu
been utilised in the form of jamu to heal various ailments. Jamu is made
is made from various herbal infusions, a combination of fresh, dried, or dried and powdered
from various herbal infusions, a combination of fresh, dried, or dried
medicinal plants, and occasionally an extract of herbs. It may compose of a single herb, but it is
and powdered medicinal plants, and occasionally an extract of herbs. It
mostly a composition of various herbs. The rhizomes of the Zingiberaceae family are commonly used
may compose of a single herb, but it is mostly a composition of various
in jamu, including temu lawak, halia, temu pauh, temu kunci, temu ireng, and others. In addition,
herbs. The rhizomes of the Zingiberaceae family are commonly used in
individual plant, such as manjakarni, kacip Fatimah, misai kucing, tongkat ali, hempedu bumi, and
jamu, including temu lawak, halia, temu pauh, temu kunci, temu ireng,
kulim are also used as herbal medicine.
Instead of verbal inheritance, these medical formulations have been passed down over
generations through documented handwriting in old manuscripts in different languages, such as Jawi
(Malay, Aceh, and Minangkabau), Primbon (Java), Paririmbon (Sunda), and Bali (Indriasari, 2011).
For example, the Jawi handwritten prescription was recorded in the Kitab Tip (medical or magical
prescription), which described the use of medicinal plants (Aswandi Syahri, 2018), while the printed
version of the manuscript had been published in the 20 century, such as in the Cabe Puyang
th
Warisan Nenek Moyang that prescribed simple and direct use of the medicinal plants (Figure 2)
(Mardisiswojo & Rajakmangunsudarso, 1985). Nowadays, commercialised traditional medicines are
available as tablets, pills, tonics, capsules, and powder form and are intended for internal use. Jamu
is also available in the form of ointments, oils, tonics, or compressed for external use (Tuschinsky,
2016).
8
4 / Drug Discovery and Development: Prospects and Challenges
and others. In addition, individual plant, such as manjakarni, kacip
Fatimah, misai kucing, tongkat ali, hempedu bumi, and kulim are also
used as herbal medicine.
Instead of verbal inheritance, these medical formulations have been
passed down over generations through documented handwriting in old
manuscripts in different languages, such as Jawi (Malay, Aceh, and
Minangkabau), Primbon (Java), Paririmbon (Sunda), and Bali (Indriasari,
2011). For example, the Jawi handwritten prescription was recorded in
the Kitab Tip (medical or magical prescription), which described the use
of medicinal plants (Aswandi Syahri, 2018), while the printed version of
the manuscript had been published in the 20 century, such as in the Cabe
th
Puyang Warisan Nenek Moyang that prescribed simple and direct use of
the medicinal plants (Figure 2) (Mardisiswojo & Rajakmangunsudarso,
1985). Nowadays, commercialised traditional medicines are available
as tablets, pills, tonics, capsules, and powder form and are intended for
internal use. Jamu is also available in the form of ointments, oils, tonics,
or compressed for external use (Tuschinsky, 2016).
Figure 2: Prescription of herbal medicine in traditional Malay medicine manuscript
Figure 2: Prescription of herbal medicine in traditional
Malay medicine manuscript
According to the Ekor (Ekor, 2014), the use of traditional, complementary, and alternative
According to Ekor (2014), the use of traditional, complementary,
medicine is increasing globally. It was reported that up to 80% of the population in low- and middle-
income countries would rely on traditional medicine for their primary healthcare needs. Interestingly,
and alternative medicine is increasing globally. It was reported that up to
complementary and alternative medicine are also becoming increasingly popular in many high-income
80% of the population in low- and middle-income countries would rely
nations with up to 65% of the population stating that they have used this type of medication. For
on traditional medicine for their primary healthcare needs. Interestingly,
example, traditional medicine is still used by up to 90% of Africans and 70% of Indians to address
complementary and alternative medicine are also becoming increasingly
their healthcare needs (Wachtel-Galor & Benzie, 2011).
popular in many high-income nations with up to 65% of the population
The main source of traditional and alternative medicines is from the virgin forest and natural
stating that they have used this type of medication. For example,
biodiversity. Tropical forests remain the main source of trees, which contributed to the global supply
of medicinal and aromatic plants (Figure 3). In the current scenario, we should focus our attention on
traditional medicine is still used by up to 90% of Africans and 70% of
the crucial yet delicate relationship between traditional medicine and modern therapy. Would we be
Indians to address their healthcare needs (Wachtel-Galor & Benzie, 2011).
able to guarantee the sustainability of traditional and alternative drug sources from nature given the
The main source of traditional and alternative medicines is from the
increasing concern over the global environmental crisis, such as deforestation and climate change?
virgin forest and natural biodiversity. Tropical forests remain the main
According to reports, the rate of deforestation between 2015 and 2020 is expected to reach 10 million
hectares per year, while primary forests have been reduced by nearly 80 million hectares owing to the
rapid agricultural growth since 1990 (FAO, 2020).
9
Drug Discovery and Development: Prospects and Challenges / 5
source of trees, which contributed to the global supply of medicinal and
aromatic plants (Figure 3). In the current scenario, we should focus our
attention on the crucial yet delicate relationship between traditional medicine
and modern therapy. Would we be able to guarantee the sustainability of
traditional and alternative drug sources from nature given the increasing
concern over the global environmental crisis, such as deforestation and
climate change? According to reports, the rate of deforestation between
2015 and 2020 is expected to reach 10 million hectares per year, while
primary forests have been reduced by nearly 80 million hectares owing to
the rapid agricultural growth since 1990 (FAO, 2020).
Figure 3: Ten countries with the most tree species (FAO, 2020)
Figure 3: Ten countries with the most tree species (FAO, 2020)
Recent studies have shown that more than 1,400 tree species are critically endangered (FAO,
Recent studies have shown that more than 1,400 tree species are
2020). Without a mitigation strategy, the main sources of traditional medicines, such as plants and
critically endangered (FAO, 2020). Without a mitigation strategy, the
their ecosystem, may face an inevitable extinction in the near future. Hence, sustainable effort must be
main sources of traditional medicines, such as plants and their ecosystem,
in line with each country's policy with the full support of the United Nations to avoid such catastrophic
may face an inevitable extinction in the near future. Hence, sustainable
loss of flora and fauna.
effort must be in line with each country’s policy with the full support of
the United Nations to avoid such catastrophic loss of flora and fauna.
Brothers and sisters,
Ladies and Gentlemen,
Brothers and sisters,
PROGRESS IN PHARMACEUTICAL DEVELOPMENT
Ladies and Gentlemen,
The development process of a new drug from the initial stage to launching a finished product
in the market is very complex, which could take 12–15 years and cost over USD 1 billion (Hughes et
Progress in Pharmaceutical Development
al., 2011). Despite its high cost and high-risk features, the global pharmaceutical market has
experienced tremendous market growth in recent years. As of the end of 2019, the pharmaceutical
The development process of a new drug from the initial stage to launching
market value was recorded at a staggering USD 1.25 trillion, which saw a significant jump from 2001
a finished product in the market is very complex, which could take 12–15
when the market value was only USD 390 billion (Mikulic, 2021). The United States remains the main
player in the global pharmaceutical market, followed by a group of emerging markets, such as Brazil,
India, Russia, Colombia, and Egypt.
The Johnson & Johnson Company gained the highest revenue in 2020 in which the
pharmaceutical division's revenues increased by 3.6%, driven by immunology (USD 13.95 billion) and
10
6 / Drug Discovery and Development: Prospects and Challenges
years and cost over USD 1 billion (Hughes et al., 2011). Despite its
high cost and high-risk features, the global pharmaceutical market has
experienced tremendous market growth in recent years. As of the end
of 2019, the pharmaceutical market value was recorded at a staggering
USD 1.25 trillion, which saw a significant jump from 2001 when the
market value was only USD 390 billion (Mikulic, 2021). The United
States remains the main player in the global pharmaceutical market,
followed by a group of emerging markets, such as Brazil, India, Russia,
Colombia, and Egypt.
The Johnson & Johnson Company gained the highest revenue in
2020 in which the pharmaceutical division’s revenues increased by 3.6%,
driven by immunology (USD 13.95 billion) and cancer (USD 10.69
billion) products. At the time of this writing, the COVID-19 vaccine is
being developed by Johnson & Johnson using vector technology and it
is scheduled for release in the first quarter of 2021 for emergency use
(Figure 4) (Pharmaceutical Technology, 2021). In terms of individual
cancer (USD 10.69 billion) products. At the time of this writing, the COVID-19 vaccine is being
drugs, Humira by Abvvie was the best-selling pharmaceutical drug in
developed by Johnson & Johnson using vector technology and it is scheduled for release in the first
2020 with USD 20.4 billion in sales. Approved in 2002 in the United
quarter of 2021 for emergency use (Figure 4) (Pharmaceutical Technology, 2021). In terms of
States, Humira is a prescription drug that is used to treat rheumatoid
individual drugs, Humira by Abvvie was the best-selling pharmaceutical drug in 2020 with USD 20.4
and psoriatic arthritis through subcutaneous injection. The drug is also
billion in sales. Approved in 2002 in the United States, Humira is a prescription drug that is used to
used to treat autoimmune diseases, such as Crohn’s disease, ankylosing
treat rheumatoid and psoriatic arthritis through subcutaneous injection. The drug is also used to treat
spondylitis, ulcerative colitis, psoriasis, uveitis, and others. Besides
autoimmune diseases, such as Crohn's disease, ankylosing spondylitis, ulcerative colitis, psoriasis,
Humira, the highest selling pharmaceutical products include Eliquis and
uveitis, and others. Besides Humira, the highest selling pharmaceutical products include Eliquis and
Revlimid, which are used to treat multiple chronic conditions or cancer
Revlimid, which are used to treat multiple chronic conditions or cancer (Figure 5) (Pharmaceutical
(Figure 5) (Pharmaceutical Technology, 2021).
Technology, 2021).
Revenue (bn)
60
50
40
30
20
10
0
Johnson & Johnson Pfizer Roche Novartis Merck & Co. GlaxoSmithKline Sanofi AbbVie Takeda Shanghai…
Figure 4: Top ten pharmaceutical companies with
the highest revenue in 2020
Figure 4: Top ten pharmaceutical companies with the highest revenue in 2020
Sales in billion U.S dollars
25
20
15
10
5
0
Stelara (Janssen Biotech)
Xarelto (Bayer/J&J)
Humira (Abbvie) Revlimid (BMS) Eylea (Bayer/Regeneron) Opdivo (BMS/Ono Pharm.) Imbruca (Abbvie) Biktarvy (Gilead)
Keytruda (Merck & Co.)
Eliquis (BMS/Pfizer)
Figure 5: The leading pharmaceutical product sales in 2020
11
cancer (USD 10.69 billion) products. At the time of this writing, the COVID-19 vaccine is being
developed by Johnson & Johnson using vector technology and it is scheduled for release in the first
quarter of 2021 for emergency use (Figure 4) (Pharmaceutical Technology, 2021). In terms of
individual drugs, Humira by Abvvie was the best-selling pharmaceutical drug in 2020 with USD 20.4
billion in sales. Approved in 2002 in the United States, Humira is a prescription drug that is used to
treat rheumatoid and psoriatic arthritis through subcutaneous injection. The drug is also used to treat
autoimmune diseases, such as Crohn's disease, ankylosing spondylitis, ulcerative colitis, psoriasis,
uveitis, and others. Besides Humira, the highest selling pharmaceutical products include Eliquis and
Revlimid, which are used to treat multiple chronic conditions or cancer (Figure 5) (Pharmaceutical
Technology, 2021).
60
50
40
30
20
10
0
GlaxoSmithKline
Johnson & Johnson Pfizer Roche Novartis Revenue (bn) Sanofi AbbVie Takeda Shanghai…
Merck & Co.
Drug Discovery and Development: Prospects and Challenges / 7
Figure 4: Top ten pharmaceutical companies with the highest revenue in 2020
Sales in billion U.S dollars
25
20
15
10
5
0
Keytruda (Merck & Co.)
Humira (Abbvie) Revlimid (BMS) Eylea (Bayer/Regeneron) Opdivo (BMS/Ono Pharm.) Imbruca (Abbvie) Biktarvy (Gilead)
Xarelto (Bayer/J&J)
Stelara (Janssen Biotech)
Eliquis (BMS/Pfizer)
Figure 5: The leading pharmaceutical product sales in 2020
Figure 5: The leading pharmaceutical product sales in 2020
Brothers and sisters,
Ladies and Gentlemen, 11
Source of Drugs
The available drugs in the market today are derived from various
sources, including natural, synthetic, biosynthetic, and recombinant
technology. The natural source of drugs includes those from plants,
animals, marines, and minerals, as well as microbiological sources.
Given its long history as an effective remedy for a variety of ailments,
plant-derived compounds have been used in the clinical setting with better
patient acceptance and tolerance. To date, about 35,000–70,000 plant
species have been tested for their medicinal use. In particular, plants that
possess ethnopharmacological properties have been the major sources
of medicine in the development of early drugs. According to Fabricant
and Farnsworth, 80% of the 122 plant-derived medicines are used for
ethnopharmacological purposes (Fabricant & Farnsworth, 2001).
When a new disease or clinical condition emerges and could not be
treated with any currently available medication, this trigger a motivation
to initiate a drug discovery project (Hughes et al., 2011). The early
stage of drug discovery begins with the ethnopharmacological approach
by random selection or chemotaxonomic approach (Figure 6). The
plant sample is obtained using conventional extraction methods, such
as maceration, percolation, soxhlet extraction, and non-conventional
8 / Drug Discovery and Development: Prospects and Challenges
extraction, such as ultrasound, microwave, enzyme-assisted extraction,
and subcritical and supercritical fluid extraction.
Figure 6: General procedure to develop a new drug from plant sources
Figure 6: General procedure to develop a new drug from plant sources
The extract is purified using chromatography methods, such as column
chromatography, preparative Thin Layer Chromatography (TLC), and
The extract is purified using chromatography methods, such as column chromatography,
High-Performance Liquid Chromatography (HPLC). Then, in vitro and
preparative Thin Layer Chromatography (TLC), and High-Performance Liquid Chromatography
in vivo bioactivities of the purified compound is tested and analysed.
(HPLC). Then, in vitro and in vivo bioactivities of the purified compound is tested and analysed.
Furthermore, enzymes and cell culture assay are used to assess the in
Furthermore, enzymes and cell culture assay are used to assess the in vitro bioactivity as well as
vitro bioactivity as well as numerous biological activities in individual
numerous biological activities in individual cells without the distractions and potential confounding
cells without the distractions and potential confounding variables that can
variables that can occur in whole organisms (Schulman, 2020). Therefore, the compiling of all
occur in whole organisms (Schulman, 2020). Therefore, the compiling
information with regard to the support on the use of traditional medicine for modern use in the
of all information with regard to the support on the use of traditional
translational study is a very important stage in drug discovery and development (Аrbаin et al., 2021).
medicine for modern use in the translational study is a very important
Most drug products fail to reach the market due to two reasons, the first is that they do not work and
the second is that they are not safe (Hughes et al., 2011).
13
Drug Discovery and Development: Prospects and Challenges / 9
stage in drug discovery and development (Аrbаin et al., 2021). Most drug
products fail to reach the market due to two reasons, the first is that they
do not work and the second is that they are not safe (Hughes et al., 2011).
Brothers and sisters,
Brothers and sisters,
Ladies and Gentlemen,
Ladies and Gentlemen,
JOURNEY IN RESEARCH: A PERSONAL EXPERIENCE
Journey in Research: A Personal Experience
Isolation and Characterisation Studies
Isolation and Characterisation Studies
We have successfully isolated and characterised several compounds from different plants
We have successfully isolated and characterised several compounds from
throughout our research journey using chromatography and spectroscopic methods. At the beginning
different plants throughout our research journey using chromatography
of the experiential research, we focused on Rubiaceae and Guttiferae families. As a young researcher,
and spectroscopic methods. At the beginning of the experiential research,
we focused on Rubiaceae and Guttiferae families. As a young researcher,
it was very exciting to work on natural products. The first impression at that time was on the
it was very exciting to work on natural products. The first impression at
Ophiorrhiza species. This species was a research interest of former supervisor Professor Dayar Arbain
that time was on the Ophiorrhiza species. This species was a research
interest of former supervisor Professor Dayar Arbain of Universitas
of Universitas Andalas. Ophiorrhiza species is a member of the Rubiaceae family. The first compound
Andalas. Ophiorrhiza species is a member of the Rubiaceae family.
that we successfully isolated is the alkaloid harman (Figure 7) from Ophiorrhiza sp. (Arbain et al.,
The first compound that we successfully isolated is the alkaloid harman
1998). Harman is a group of indole alkaloids originally isolated from Peganum harmala. Harman is
(Figure 7) from Ophiorrhiza sp. (Arbain et al., 1998). Harman is a group
of indole alkaloids originally isolated from Peganum harmala. Harman
reported to exhibit strong anti-depressant activity (Ferraz et al., 2019).
is reported to exhibit strong anti-depressant activity (Ferraz et al., 2019).
Figure 7: Alkaloid harman compound from Ophiorrhiza sp.
Figure 7: Alkaloid harman compound from Ophiorrhiza sp.
In our recent review of 50 Ophiorrhiza species, we found that
Ophiorrhiza has significant biological activities. The plants have been
In our recent review of 50 Ophiorrhiza species, we found that Ophiorrhiza has significant
used traditionally to treat pain, inflammation, cancer, and bacterial-
biological activities. The plants have been used traditionally to treat pain, inflammation, cancer, and
and viral-based infections. In addition, Ophiorrhiza species can treat
snakebite, stomatitis, ulcers, and wounds and function as an antioxidant,
bacterial- and viral-based infections. In addition, Ophiorrhiza species can treat snakebite, stomatitis,
antitussive, and analgesic. They are also used to treat gastropathy, leprosy,
ulcers, and wounds and function as an antioxidant, antitussive, and analgesic. They are also used to
and amenorrhea and have sedative and laxative properties that can be
treat gastropathy, leprosy, and amenorrhea and have sedative and laxative properties that can be
extracted from their root barks. Overall, the Ophiorrhiza species possess a wide range of biological
activities and phytochemical constituents, as shown in Figure 8 (Krishnan et al., 2014; Midhu et al.,
2019; Taher et al., 2020).
14
10 / Drug Discovery and Development: Prospects and Challenges
extracted from their root barks. Overall, the Ophiorrhiza species possess
a wide range of biological activities and phytochemical constituents, as
shown in Figure 8 (Krishnan et al., 2014; Midhu et al., 2019; Taher et
al., 2020).
Figure 8: Summary of the biological activities and phytochemicals
compounds in Ophiorrhiza species
Campthothecin was originally found in the Chinese tree Camptotheca
acuminata, which was later found in several Ophiorrhiza species, such as
O. mungos, O. mungos var. angustifolia, and O. rugosa var. decumbens.
Due to its limited water solubility and significant toxicity during the
S-phase of the cell cycle that can result in normal cell death, clinical
studies on camptothecin were unsuccessful, which makes camptothecin
unable to penetrate the worldwide market (Li et al., 2017).
Nonetheless, the potential anti-cancer properties of camptothecin
have led researchers to develop several other camptothecin analogues,
such as topotecan (topotecan injection, Hycamtin and Potactasol ) for
®
®
ovarian cancer that has spread to other parts of the body and irinotecan
(irinotecan injection, Camptosar ), which is used to treat colon cancer
®
and small-cell lung cancer. These chemical analogues demonstrated
better DNA-topoisomerase-1 inhibitor activities and are well-tolerated
compared to the natural camptothecin.
Drug Discovery and Development: Prospects and Challenges / 11
The high demand for camptothecin analogues in the market from
USD 3,500 to USD 50,000 per kilogram has inspired researchers to
produce its callus culture. The production of camptothecin analogues
has been developed using the plant culture propagation technique. In
vitro propagation of O. mungos recorded the production of camptothecin
compared to naturally grown O. mungos, proving that the culture
propagation strategy was successful (Namdeo et al., 2012). Figure 9
shows the strategic model implemented in drug discovery from plants
O. mungos recorded the production of camptothecin compared to naturally grown O. mungos, proving
that the culture propagation strategy was successful (Namdeo et al., 2012). Figure 9 shows the
for unfavourable metabolites.
strategic model implemented in drug discovery from plants for unfavourable metabolites.
Figure 9: Model of drug discovery and development of
Figure 9: Model of drug discovery and development of new drugs through plant culture technique
new drugs through plant culture technique
Our research interest also focused on the Guttiferae family. The Guttiferae family is a tropical
Our research interest also focused on the Guttiferae family. The
plant with four major genera comprising Garcinia, Calophyllum, Mesua, and Mammea. The Guttiferae
Guttiferae family is a tropical plant with four major genera comprising
family is known to possess unique secondary metabolites, such as xanthones (rubraxanthones, a-
mangostin, and b-mangostin) (Jantan et al., 2002; Taher et al., 2006, 2015), triterpenoids, and
Garcinia, Calophyllum, Mesua, and Mammea. The Guttiferae family
coumarins. We have also successfully isolated a new compound called enervosanone, which is a
is known to possess unique secondary metabolites, such as xanthones
polyisoprenylated ketone, from Callopyhlum enervosum (Taher et al., 2005) and biscaloxanthone, a
(rubraxanthones, a-mangostin, and b-mangostin) (Jantan et al., 2002;
xanthone dimer from C. canum (Taher et al., 2021) (Figure 10).
Taher et al., 2006, 2015), triterpenoids, and coumarins. We have also
successfully isolated a new compound called enervosanone, which is a
polyisoprenylated ketone, from Callopyhlum enervosum (Taher et al.,
2005) and biscaloxanthone, a xanthone dimer from C. canum (Taher et
al., 2021) (Figure 10).
16
12 / Drug Discovery and Development: Prospects and Challenges
O OH
Field Code Changed
Field Code Changed
O OH
O O O OH O O OH
O O HO O OH O O OH
O HO O O
O
O O
Figure 10: Enervosanone and biscaloxanthone isolated from
C. enervosum and C. canum, respectively
Figure 10: Enervosanone and biscaloxanthone isolated from C. enervosum and C. canum,
The most popular plant from the genus Calophyllum is the rare C.
respectively
lanigerum var austrocoriaceum found in Sarawak. The C. laginerum
has gained special attraction due to its promising activity against type 1
The most popular plant from the genus Calophyllum is the rare C. lanigerum var
Human Immunodeficiency Virus (HIV-1). The novel calanolide A, which
austrocoriaceum found in Sarawak. The C. laginerum has gained special attraction due to its promising
is a naturally occurring, non-nucleoside reverse transcriptase inhibitor
activity against type 1 Human Immunodeficiency Virus (HIV-1). The novel calanolide A, which is a
was found to be active in vitro. In addition, the coumarin moiety in
naturally occurring, non-nucleoside reverse transcriptase inhibitor was found to be active in vitro. In
the calanolide A structure (Figure 11) has a wide range of biological
addition, the coumarin moiety in the calanolide A structure (Figure 11) has a wide range of biological
activities, including anti-bacterial, anti-cancer, anti-coagulant, anti-
activities, including anti-bacterial, anti-cancer, anti-coagulant, anti-fungal, and anti-viral (Xu et al.,
fungal, and anti-viral (Xu et al., 2021). Many coumarin-based drugs
2021). Many coumarin-based drugs have been introduced in the clinical setting, such as warfarin.
have been introduced in the clinical setting, such as warfarin. Calanolide
Calanolide A is currently under clinical trial to evaluate the activity of coumarin derivatives as anti-
A is currently under clinical trial to evaluate the activity of coumarin
HIV agents to treat HIV-infected patients.
derivatives as anti-HIV agents to treat HIV-infected patients.
Figure 11: Structure of the coumarin skeleton and calanolide A
Figure 11: Structure of the coumarin skeleton and calanolide A from C. laginerum
from C. lanigerum
17
Drug Discovery and Development: Prospects and Challenges / 13
Brothers and sisters,
Ladies and Gentlemen,
In Vitro and In Vivo Studies
In the initial step of drug discovery, all potential lead compounds
undergo in vitro pharmacology testing. Various preliminary testing
is available, including chemical reactions, enzymes reaction, and
cell/ microbial culture tests. In vitro studies using chemical reactions,
such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis-(3-
ethylbenzothiazoline-6-sulfonate (ABTS), and lipid peroxidation are
normally used for the antioxidant assay. In addition, enzyme assays,
such as a-glucosidase and a-amylase assays, are conducted for anti-
diabetic testing.
The cell line experiment is an important step to analyse in vitro
pharmacology activity, which explains the mechanism of action of
the drug in detail, including protein and gene expression, as well as
a cytotoxic activity but it cannot fully replace animal testing in the
drug development process. This is because in vitro studies lack certain
information regarding substance safety, toxicity, pharmacokinetics, and
metabolic pathway.
There is a wide range of cells that are available for bioassay testing,
such as the RAW 264.7 cell lines for anti-inflammatory assay. In this
study, the murine RAW 264.7 cells were induced with pro-inflammatory
Lipopolysaccharide (LPS). LPS plays an important role in inducing the
inflammatory, which leads to various inflammatory diseases, and it is a
compound of the cellular wall of Gram-negative bacteria (Takeuchi &
Akira, 2010). The formation of nitric oxide (NO) produced by the cells
was determined using the Griess reagent. It was postulated that excessive
production of NO plays a critical role in the pathogenesis of inflammation
(Bahiense et al., 2017). This method showed a clear response of RAW
264.7 macrophages towards the inflammatory agent by producing NO,
which will be inhibited by an anti-inflammatory agent. The following
sections describe our previous and on-going research studies on drug
discovery and development, which involves anti-cancer/cytotoxicity,
anti-hyperglycemic, anti-obesity, and wound healing.
14 / Drug Discovery and Development: Prospects and Challenges
Brothers and sisters,
Ladies and Gentlemen,
Anti-cancer/Cytotoxicity
Various cytotoxic and anti-cancer studies have used 4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) and its improved MTS
assay. Firstly introduced by Mossman in 1983, the MTT assay uses
mitochondrial reductase enzymes produced by live cells to convert MTT
to yellow formazan (Figure 12). This method is very useful to evaluate
the cytotoxic effect of lead compounds and determine the half maximal
inhibition concentration (IC50) of the compounds. MTT results provide
important information with regards to the concentration of the sample
to be used in the experiment and to evaluate the biocompatibility of
material in the formulation of newly introduced polymers. Recently, a
cytotoxic study was conducted with brine shrimp lethality assay before
the introduction of cell culture in drug discovery. Different types of
cancer cells, such as A549, Caco-2, and H1299, have become popular
as an initial screening for anti-cancer activity.
Figure 12: MTT assay of b-mangostin on 96-well plate
In one of our anti-cancer studies, we discovered that b-mangostin
inhibited the growth of the tested cell lines. Due to the limited
physicochemical properties of b-mangostin, we are planning to formulate
b-mangostin in PEGylated liposome to improve its delivery (Taher, 2021
Drug Discovery and Development: Prospects and Challenges / 15
unpublished). Another study showed that triterpenoids from Luvunga
scandens inhibited breast cancer cells (Al-Zikri et al., 2014). To extend
our research, we have collaborated with other Kulliyah to study marine-
based products. One of the studies was focused on sea cucumber and
seaweed (Ruslan, et al., 2021). Sea cucumbers, such as Holothuria edulis
Lesson and Stichopus horrens Selenka, were reported to possess potent
cytotoxic activities (Althunibat et al., 2013).
Brothers and sisters,
Ladies and Gentlemen,
Anti-hyperglycemic
Insulin resistance is the main precursor of non-insulin-dependent diabetes
mellitus (type 2 diabetes). Insulin resistance is characterised by reduced
insulin-stimulated glucose absorption in muscle and adipose tissue and
impaired insulin-suppressed glucose production in the liver due to obesity
(Reaven, 1988). According to the International Diabetes Federation
(IDF), the number of diabetic people in the population is projected to
increase to 578 million by 2030 (International Diabetes Federation, 2021)
(International Diabetes Federation, 2021).
A recent study reported that over 800 plant species have been proven
to treat diabetes, which is mostly based on various in vitro study models
and animal testing (Furman et al., 2020). In our research, we also applied
the cell culture method for anti-diabetes and anti-obesity assays using
3T3-L1 preadipocytes cells. Preadipocyte cells need to be induced with
a chemical cocktail to become adipocytes cells. The cocktail consists of
3-isobutyl-1-methylxanthine (IBMX), dexamethasone, and insulin. The
stimulation process takes place from 8 to 14 days to allow the adipocyte
to reach maturity (Table 1 and Figure 13). The proliferation of adipose
tissue comprises the expansion of mature adipocytes and the production
of new adipocytes from locally resident preadipocytes (Lai et al., 2012).
The formation of lipid droplets (Figure 14) indicates that the adipocyte
cells have undergone a differentiation process. This stage allows us to
manipulate or test their response toward the treatment.
16 / Drug Discovery and Development: Prospects and Challenges
Table 1: Cocktail used in adipocyte differentiation study
Negative Positive Positive control 2 Treatment 1
Day
control control 1 (Rosiglitazone) (Sample X)
0 Complete Dexamethasone Dexamethasone Dexamethasone
DMEM (0.25 µM) (0.25 µM) (0.25 µM)
IBMX (0.5 mM) IBMX (0.5 mM) IBMX (0.5 mM)
Insulin (10 µg/mL) Insulin (10 µg/mL) Insulin (10 µg/mL)
Complete DMEM Rosiglitazone Sample X (10, 45,
Complete DMEM and 60 μg/ mL)
Complete DMEM
2 Complete Insulin (10 µg/mL) Insulin (10 µg/mL) Insulin (10 µg/mL)
DMEM Complete DMEM Rosiglitazone Sample X (10, 45,
Complete DMEM and 60 μg/ mL)
Complete DMEM
4 Complete Complete DMEM Complete DMEM Rosiglitazone Sample X (10, 45,
Rosiglitazone
Sample X (10, 45,
Complete DMEM
and 60 µg/ mL)
DMEM Complete DMEM and 60 μg/ mL)
Complete DMEM
4 Complete DMEM Complete DMEM Rosiglitazone Sample X (10, 45,
Complete DMEM
6 Complete Complete DMEM Rosiglitazone Complete DMEM and 60 µg/ mL)
Sample X (10, 45,
Complete DMEM
Complete DMEM
6 DMEM Complete DMEM Complete DMEM Rosiglitazone Sample X (10, 45,
and 60 μg/ mL)
Complete DMEM and 60 µg/ mL)
Complete DMEM
Sample X (10, 45,
Complete DMEM Complete DMEM
8 8 Complete Complete DMEM Rosiglitazone Rosiglitazone Complete DMEM
Sample X (10, 45,
Complete DMEM
DMEM Complete DMEM and 60 μg/ mL)
and 60 µg/ mL)
Complete DMEM
Complete DMEM
Figure 13: Simplified procedure of in vitro diabetes study using cell culture
Figure 13: Simplified procedure of in vitro diabetes study using cell culture
21
Drug Discovery and Development: Prospects and Challenges / 17
Figure 14: Mature adipocyte stained with oil red O
The effects of several phytochemical compounds have been
studied on 3T3-L1 adipocytes, such as cinnamtannin B1, which is
a type of proanthocyanidin from Cinnamomum zeylanicum (Figure
15). Cinnamtannin B1 showed an improved glucose uptake in 3T3-L1
preadipocytes. In our study, we have concluded that the sweet agent
cinnamtannin B1 in cinnamon mimics insulin by attaching to the β-subunit
of the receptor in the cell membrane, followed by autophosphorylating
the tyrosine residues of the b-subunit. Subsequently, the phosphoinositide
3-kinases (PI3K), glucose transporter 4 (GLUT4) translocation, and
glucose absorption were activated. Based on the response of the
cinnamtannin B1, it was concluded that the phosphorylation of the
insulin receptor (IR ) b-subunit is an important molecular target for the
treatment of diabetes (Taher et al., 2004, 2006, 2007).
Fascinatingly, our finding had attracted the attention of the national
newspaper at that time with an article “Sweet hope for diabetics”
(Abdullah, 2006). The study was supported by a clinical trial on aqueous
cinnamon extract in which 120 mg (low dose) and 360 mg (high dose)
mg was given to patients with type 2 diabetes daily for three months.
The results showed that the HbA1c and fasting plasma glucose levels
significantly decreased (reduction of 1.62 mmol/L vs. 0.22 mmol/L in
the placebo group) (Lu et al., 2012). Based on a systematic review and
meta-analysis studies involving 1025 participants, the supplementation
of cinnamon was found to decrease serum triglycerides, total cholesterol,
and low-density lipoprotein cholesterol (Jamali et al., 2020).
Figure 14: Mature adipocyte stained with oil red O
The effects of several phytochemical compounds have been studied on 3T3-L1 adipocytes,
such as cinnamtannin B1, which is a type of proanthocyanidin from Cinnamomum zeylanicum (Figure
15). Cinnamtannin B1 showed an improved glucose uptake in 3T3-L1 preadipocytes. In our study, we
have concluded that the sweet agent cinnamtannin B1 in cinnamon mimics insulin by attaching to the
β-subunit of the receptor in the cell membrane, followed by autophosphorylating the tyrosine residues
of the β-subunit. Subsequently, the phosphoinositide 3-kinases (PI3K), glucose transporter 4 (GLUT4)
translocation, and glucose absorption were activated. Based on the response of the cinnamtannin B1,
it was concluded that the phosphorylation of the insulin receptor (IR )β-subunit is an important
molecular target for the treatment of diabetes (Taher et al., 2004, 2006, 2007).
Fascinatingly, our finding had attracted the attention of the national newspaper at that time with
an article “Sweet hope for diabetics” (Abdullah, 2006). The study was supported by a clinical trial on
aqueous cinnamon extract in which 120 mg (low dose) and 360 mg (high dose) mg was given to
patients with type 2 diabetes daily for three months. The results showed that the HbA1c and fasting
plasma glucose levels significantly decreased (reduction of 1.62 mmol/L vs. 0.22 mmol/L in the
placebo group) (Lu et al., 2012). Based on a systematic review and meta-analysis studies involving
1025 participants, the supplementation of cinnamon was found to decrease serum triglycerides, total
18 / Drug Discovery and Development: Prospects and Challenges
cholesterol, and low-density lipoprotein cholesterol (Jamali et al., 2020).
R=H is a cinnamtannin B1, R=acetate is an acetate derivative
Figure 15: Cinnamtannin B1 and its acetate derivative
22
The α-mangostin from Garcinia mangostana also demonstrated a
significant in vitro anti-hyperglycemic effect. In comparison to cells
treated with 0.5 mM IBMX, 0.25 mM dexamethasone, and 1 g/mL insulin
(MDI), cells treated with a-mangostin (50 M) recorded a substantial
reduced intracellular lipid accumulation in a dose-dependent manner
of up to 44.4%. The uptake activity of 2-deoxy-D-[3H] glucose was
determined to be significantly increased by α-mangostin (p < 0.05) with
the maximum activity at 25 μM. Furthermore, α-mangostin increased
the amount of Free Fatty Acids (FFA) secreted by the cells (Taher et
al., 2015). A new lignan, isocubebinic ether from Knema patentinervia
showed promising activity in adipocyte differentiation. It was found that
the compound significantly improved the glucose uptake (p < 0.05) in
3T3-L1 adipocytes at 50 mg/mL (Taher et al., 2017).
Triterpenoids, friedelin, and lanosterol from G. prainiana (Susanti
et al., 2013) were found to exhibit mimicking insulin activities by
stimulating fat accumulation (2.02 and 2.18 folds for triterpenoids and
lanosterol, respectively), while friedelin stimulates glucose uptake by 1.8
fold. A recent study by Sunil et al (2021) showed that friedelin enhanced
the translocation and activation of GLUT2 and GLUT4 through the
activation of PI3K/p-Akt signalling cascade in skeletal muscles and liver
of diabetic rats (Sunil et al. 2021).
The in vitro anti-diabetic and anti-obesity studies using 3T3-
L1 adipocytes are gaining popularity due to their ability to provide
preliminary information regarding glucose metabolism activity.
Adipocyte cells play an important role in improving our understanding
of systemic metabolic homeostasis. Knowledge of the physiology of
adipose tissue provides the foundation for a mechanistic understanding
Drug Discovery and Development: Prospects and Challenges / 19
of diabetes pathogenesis (Scherer, 2019). Furthermore, studies on
adipocyte cells allow us to understand the cellular response toward
glucose metabolism. Therefore, the mechanism of action of the molecule
on glucose regulation could be predicted. The parameters that provide a
significant understanding of the mechanism of glucose metabolism are
concerning protein and gene expression by adipocytes with regard to
glucose transporter and insulin receptor regulation (Figure 16).
Figure 16: The role of adipocyte in glucose transport (Biorender.com)
Figure 16: The role of adipocyte in glucose transport (Biorender.com)
Brothers and sisters,
Brothers and sisters,
Ladies and Gentlemen,
Ladies and Gentlemen,
Anti-obesity Anti-obesity
The global rising incidence of obesity is becoming a major concern in public health. Obesity leads to
The global rising incidence of obesity is becoming a major concern in
the development of metabolic disorders such as diabetes, hypertension and cardiovascular diseases
public health. Obesity leads to the development of metabolic disorders
(Mohamed et al., 2014). Nowadays, many conventional drugs have been used to manage obesity. The
harmful effect and availability of these drugs initiates the researchers to find the alternatives including
such as diabetes, hypertension and cardiovascular diseases (Mohamed
from plants. It was revealed that the gelugor fruit, G. atroviridis contains Hydroxycitric Acid (HCA)
et al., 2014). Nowadays, many conventional drugs have been used
(Figure 17), which is a substance known to have obesity prevention and treatment as well as
to manage obesity. The harmful effect and availability of these drugs
preventing obesity-related diseases. The study estimated that 11.35 ± 0.55% (w/w) of HCA was present
initiates the researchers to find the alternatives including from plants. It
in the fruit extract (Susanti et al., 2020). The adipogenesis inhibition effect by the HCA-rich extract
was revealed that the gelugor fruit, G. atroviridis contains Hydroxycitric
on 3T3l-L1 adipocytes cells showed the modulation of C/EBPα protein expression. HCA also
Acid (HCA) (Figure 17), which is a substance known to have obesity
enhanced the adipocyte breakdown (adipolysis) in the study.
prevention and treatment as well as preventing obesity-related diseases.
The study estimated that 11.35 ± 0.55% (w/w) of HCA was present
in the fruit extract (Susanti et al., 2020). The adipogenesis inhibition
effect by the HCA-rich extract on 3T3l-L1 adipocytes cells showed
the modulation of C/EBPα protein expression. HCA also enhanced the
adipocyte breakdown (adipolysis) in the study.
24
20 / Drug Discovery and Development: Prospects and Challenges
Figure 17: Fruit of G. atroviridis and its active compound, HCA
Figure 17: Fruit of G. atroviridis and its active compound, HCA
Brothers and sisters,
Brothers and sisters,
Ladies and Gentlemen,
Ladies and Gentlemen,
Wound Healing Wound Healing
In vitro wound healing has been established using human skin fibroblast cells. In our previous
In vitro wound healing has been established using human skin fibroblast
study, Centella asiatica contains the active compound asiaticoside, which is an active compound in
cells. In our previous study, Centella asiatica contains the active
wound healing in Human Dermal Fibroblast (HDF) and Human Dermal Keratinocyte (HaCaT) (Aziz
compound asiaticoside, which is an active compound in wound healing
et al. 2017). In addition, cell culture models using HaCaT cells observed the mechanism of wound
in Human Dermal Fibroblast (HDF) and Human Dermal Keratinocyte
healing by observing cell migration under a microscope (Figure 18) (Azis et al., 2015). A thorough
description of our study on C. asiatica is described in further sections.
(HaCaT) (Aziz et al. 2017). In addition, cell culture models using HaCaT
cells observed the mechanism of wound healing by observing cell
migration under a microscope (Figure 18) (Azis et al., 2015). A thorough
description of our study on C. asiatica is described in further sections.
25
Figure 18: Wound healing activity of C. asiatica extracts on HaCaT cell lines
Figure 18: Wound healing activity of C. asiatica extracts on HaCaT cell lines (Azis et al., 2015)
(Azis et al., 2015)
26
Drug Discovery and Development: Prospects and Challenges / 21
Brothers and sisters,
Ladies and Gentlemen,
Brothers and sisters,
Ladies and Gentlemen,
Drug Development Process
DRUG DEVELOPMENT PROCESS
According to the Food and Drug Administration (FDA), drug discovery
According to the Food and Drug Administration (FDA), drug discovery and development
and development involve five stages: discovery and development, pre-
involve five stages: discovery and development, pre-clinical research, clinical research, FDA review,
clinical research, clinical research, FDA review, and post-marketing
and post-marketing surveillance (US FDA, 2020). The concise step is provided in Figure 19.
surveillance (US FDA, 2020). The concise step is provided in Figure 19.
POST
MARKETING
Figure 19: Drug discovery and development chart
Figure 19: Drug discovery and development chart
The drug development process began in university laboratories, where research groups are s,
The drug development process began in university laboratorie
where research groups are awarded research grants from government
awarded research grants from government agencies or pharmaceutical industries to carry out
agencies or pharmaceutical industries to carry out fundamental research
fundamental research to understand a particular disease. Once the potential target has been identified,
to understand a particular disease. Once the potential target has been
experimental procedures and lab testing are performed. Historically, researchers would identify
identified, experimental procedures and lab testing are performed.
potential compounds from natural sources, such as plant, animal, microbes, and marine products. As
Historically, researchers would identify potential compounds from
many as 10,000 compounds are found to exhibit potential biological activities for drug application.
natural sources, such as plant, animal, microbes, and marine products.
The process continues to progress to a pre-clinical study using in vitro and in vivo techniques, resulting
As many as 10,000 compounds are found to exhibit potential biological
in less than five compounds to proceed with the clinical trial. After a long and laborious journey, only
activities for drug application. The process continues to progress to a
one drug candidate will pass the FDA review before its official release into the market (Torjesen,
2015). pre-clinical study using in vitro and in vivo techniques, resulting in less
than five compounds to proceed with the clinical trial. After a long and
laborious journey, only one drug candidate will pass the FDA review
before its official release into the market (Torjesen, 2015).
27
22 / Drug Discovery and Development: Prospects and Challenges
Brothers and sisters,
Ladies and Gentlemen,
Importance of Animal Study
Even though the use of animals for laboratory testing is strictly regulated
with the 3R rules, namely reduction, refinement, replacement, in vivo
studies using animal testing is an important step in drug discovery.
Various organisations, such as the Organisation for Economic Co-
operation and Development (OECD), the National Institutes of Health
(NIH), the International Conference on Harmonisation of Technical
Requirements for Registration of Pharmaceuticals for Human Use
(ICH), and the Committee for the Purpose of Control and Supervision
of Experiments on Animals (CPCSEA) provide guidelines for animal
housing, breeding, feeding, and transportation (Rollin, 2003). Generally,
in vivo studies involving the use of animals, including zebrafish, birds,
mice, rats, rabbits, dogs, pigs, and even primates. In vivo studies provide
researchers with results that are closely associated with humans due to
the complexity of the organism (Doke & Dhawale, 2015).
In our study, we used streptozotocin-induced diabetes rats for the
study of the anti-hyperglycemic effect of a-mangostin and rabbits in
wound healing (Azis et al., 2017; Ahmed et al., 2019; Taher et al., 2016).
In another study, the anti-hyperglycemic effect of G. mangostana extract
has been tested on male Sprague-Dawley rats. G. mangostana extract
(GME) caused hypoglycaemia by increasing the number of insulin-
producing-cells in the body. The presence of antioxidant-bearing tannins,
such as epicatechin and xanthones, such as a -mangostin, could explain
this effect (Taher et al., 2016). Furthermore, the wound healing properties
of C. asiatica extract in the form of hydrogel formulation enhanced
the wound healing process in rabbits 15% faster than commercial
wound healing products. The healing process was characterised by the
formation of a thick epithelial layer, keratin, and mild granulation tissues,
fibroblasts, and collagen production with no fibrinoid necrosis (Ahmed
et al., 2019).
Drug Discovery and Development: Prospects and Challenges / 23
Brothers and sisters,
Ladies and Gentlemen,
Drug Formulation
Formulation is an important step in drug discovery. It provides a material
for preclinical pharmacokinetic, efficacy and toxicological studies
(Strickley, 2008). It is widely acknowledged that new medications would
exhibit certain formulation constraints, such as solubility problems. The
Biopharmaceutical Classification System (BCS) is an experimental
technique to determine the permeability and solubility of a particular
product under controlled settings. Based on their aqueous solubility
and intestinal permeability, the drugs are classified into four classes.
In addition to the BCS classification, the input from pre-formulation
studies would benefit the formulation by providing a detailed solubility
profile, polymorph status, intended dosage form, target dose, dosing
schedule, drug stability, excipient compatibility, and understanding of
the transporter and metabolic pathways.
Drugs in BCS are classified as high permeability but low solubility.
In order to improve the performance of these drugs, a special technique
is required to increase the surface area, such as particle size reduction,
using a solid solution, or solid dispersion. Nowadays, new technologies
are available to modify the formulation using solvents and/or surfactants.
One of the strategies is to encapsulate the drugs with Nanotechnology
Drug Delivery System (NDDS) (Figure 20). NDDS refers to a material
that has at least one dimension that falls under the nanometer scale (1–100
nm) or is made with basic units in three-dimensional space (Deng, 2020).
Nanotechnology has become a useful tool to regulate the delivery rate
and target the drug delivery to a specific location, such as using vesicle
nanocarrier, for instance, liposomes and niosomes. Liposome comprises
cholesterol and phospholipid, while niosome uses cholesterol and non-
ionic surfactant. The active principle in liposomes and niosome involves
the encapsulation of the drug in vesicle components of hydrophilic and
hydrophobic areas. Niosome (Figure 21) is made up of bilayer structures
and formed by self-association of non-ionic surfactant and cholesterol
Biopharmaceutical Classification System (BCS) is an experimental technique to determine the
permeability and solubility of a particular product under controlled settings. Based on their aqueous
solubility and intestinal permeability, the drugs are classified into four classes. In addition to the BCS
classification, the input from pre-formulation studies would benefit the formulation by providing a
detailed solubility profile, polymorph status, intended dosage form, target dose, dosing schedule, drug
stability, excipient compatibility, and understanding of the transporter and metabolic pathways.
Drugs in BCS are classified as high permeability but low solubility. In order to improve the
performance of these drugs, a special technique is required to increase the surface area, such as particle
size reduction, using a solid solution, or solid dispersion. Nowadays, new technologies are available
to modify the formulation using solvents and/or surfactants. One of the strategies is to encapsulate the
drugs with Nanotechnology Drug Delivery System (NDDS) (Figure 20). NDDS refers to a material
that has at least one dimension that falls under the nanometer scale (1–100 nm) or is made with basic
units in three-dimensional space (Deng, 2020).
Nanotechnology has become a useful tool to regulate the delivery rate and target the drug
delivery to a specific location, such as using vesicle nanocarrier, for instance, liposomes and niosomes.
Liposome comprises cholesterol and phospholipid, while niosome uses cholesterol and non-ionic
surfactant. The active principle in liposomes and niosome involves the encapsulation of the drug in
vesicle components of hydrophilic and hydrophobic areas. Niosome (Figure 21) is made up of bilayer
24 / Drug Discovery and Development: Prospects and Challenges
structures and formed by self-association of non-ionic surfactant and cholesterol in an aqueous phase.
They are biocompatible, biodegradable, non-immunogenic, and have a long shelf life, exhibit high
in an aqueous phase. They are biocompatible, biodegradable, non-
stability. Moreover, niosomes enable the delivery of drugs at the target site in a controlled and/or
immunogenic, and have a long shelf life, exhibit high stability. Moreover,
sustained manner (Mahale et al., 2012).
niosomes enable the delivery of drugs at the target site in a controlled
and/or sustained manner (Mahale et al., 2012).
Figure 20: Types of micro- and nanosized design for drug delivery (Biorender.com)
Figure 20: Types of micro- and nanosized design for drug delivery
(Biorender.com)
29
Figure 21: Structure of niosome
Figure 21: Structure of niosome
One of the most widely used polymers in the design and formulation
One of the most widely used polymers in the design and formulation of drug delivery systems
of drug delivery systems for biomedical purposes is Poly-Lactic-co-
for biomedical purposes is Poly-Lactic-co-Glycolic Acid (PLGA) (Ghitman et al., 2020). The FDA
Glycolic Acid (PLGA) (Ghitman et al., 2020). The FDA has approved
PLGA given its biodegradability, biosafety, biocompatibility, formulation
has approved PLGA given its biodegradability, biosafety, biocompatibility, formulation versatility,
versatility, and excellent functionalisation (Pandita et al., 2015).
and excellent functionalisation (Pandita et al., 2015). Previously, the solvent evaporation approach
Previously, the solvent evaporation approach was used to encapsulate
was used to encapsulate α-mangostin in PLGA microspheres although the entrapment efficiency was
α-mangostin in PLGA microspheres although the entrapment efficiency
low. Considering that α-mangostin is soluble in organic solvent due to its hydrophobic nature, the
microsphere formulation could be a potential α-mangostin micro-carrier delivery system to treat lung
tissue cancer (Ali et al., 2013).
Although oral intake is the most preferable route of drug delivery, more than 70% of the active
pharmaceutical ingredients are poorly water-soluble or lipophilic compounds, which prevent them
from reaching the desired efficacy. As a result, the bioavailability of poorly soluble drugs is
significantly affected. These compounds are typically grouped as BCS class II or class IV compounds
(Anwer et al., 2021). To improve the rate of absorption and solubility, the Self-Nanoemulsifying Drug
Delivery System (SNEDDS) was developed as an alternative formulation method. SNEDDS is a
relatively new emulsion technology with the purpose to improve the rate and extent of absorption of
poorly water-soluble drugs. SNEDDS are anhydrous homogeneous liquid mixtures composed of
30
Drug Discovery and Development: Prospects and Challenges / 25
was low. Considering that α-mangostin is soluble in organic solvent
due to its hydrophobic nature, the microsphere formulation could be a
potential α-mangostin micro-carrier delivery system to treat lung tissue
cancer (Ali et al., 2013).
Although oral intake is the most preferable route of drug delivery,
more than 70% of the active pharmaceutical ingredients are poorly water-
soluble or lipophilic compounds, which prevent them from reaching the
desired efficacy. As a result, the bioavailability of poorly soluble drugs
is significantly affected. These compounds are typically grouped as BCS
class II or class IV compounds (Anwer et al., 2021). To improve the rate
of absorption and solubility, the Self-Nanoemulsifying Drug Delivery
System (SNEDDS) was developed as an alternative formulation method.
SNEDDS is a relatively new emulsion technology with the purpose to
improve the rate and extent of absorption of poorly water-soluble drugs.
SNEDDS are anhydrous homogeneous liquid mixtures composed of
lipids, surfactants, drugs, and/or cosolvents, which spontaneously form
transparent and stable microemulsion upon aqueous dilution with gentle
agitation (Buya et al., 2020).
Currently, several products of SNEDDS in the market include
Aptivus (tipranavir), Kaletra (lopinavir and ritonavir), Sandimmune
Neoral (cyclosporine A), Norvir (ritonavir) and Fortovase (saquinavir).
Our recent study on SNEDDS was successful in improving the anti-
diabetic properties of andrographolide by enhancing the regeneration
of pancreatic β-cells, lowering the blood glucose levels, and inhibiting
lipid formation in adipocytes (Syukri et al., 2020).
Furthermore, raloxifene is a selective oestrogen receptor modulator
that possesses poor bioavailability even though it is useful in the treatment
of osteoporosis and potential use in breast cancer treatment. The drug has
been formulated in lipid nanocarriers, transfersome, and ethosomes using
span-80, span-85, and sodium deoxycholate, respectively. It was found
that the formulation increased the bioavailability of transfersomes and
ethosomes by 247 and 157 times, respectively (Syed Mahmood, 2017).
We conducted another study on aceclofenac using a proniosome
vesicle. Aceclofenac is a potent non-steroidal anti-inflammatory drug
that is used to relieve pain and inflammation in osteoarthritis, rheumatoid
arthritis, and ankylosing spondylitis. However, the drug has limited
use due to its low bioavailability. When the drug was formulated in
26 / Drug Discovery and Development: Prospects and Challenges
maltodextrin proniosomes, the drug exhibited fast absorption with
183% relative bioavailability compared to the pure drug, while glucose
proniosomes demonstrated 112% relative bioavailability (Sammour et
al., 2019).
PEGylation can be used to increases the size and molecular weight
of proteins and peptides while also improving their pharmacokinetics
and pharmacodynamics by increasing their water solubility, therefore,
protecting them from enzymatic degradation, lowering renal clearance,
and decreasing the immunogenic and antigenic responses (Milla et al.,
2011). The Doxil 5 (PEGylated liposomal doxorubicin) was the first
®
clinically approved liposomal nanomedicine by the FDA for the treatment
of advanced ovarian cancer, multiple myeloma, and HIV-associated
Kaposi sarcoma (Gkionis et al., 2020).
Cancer is characterised by the heterogeneous proliferation of cells
with the intrinsic potential to metastasise to major organs (Li et al., 2010).
Multidrug resistance refers to the process in which cancer cells develop
resistance against a variety of anti-cancer treatments that are structurally
and functionally distinct from the initial drug after being exposed to it.
The resistance is associated with drug efflux and metabolism, whether
it is intrinsic or acquired (Szakács et al., 2006).
A multiagent-mechanistic treatment method using free drug
cocktail treatment alternatives is used to treat such cancers. Previously,
the medication combination of doxorubicin hydrochloride (DOX)
and vincristine sulfate (VCR) was successful in treating aggressive
adenocarcinomas (Mokhtari et al., 2017). The two most significant
approval of the combination of drug nanocarrier includes Vyxeos™ and
chimeric antigen receptor T cells (CAR-T) therapy, Kymriah™ and or
Yescarta™ (Ghosh et al., 2019).
The b-mangostin is an active compound from G. mangostana with
low solubility due to its hydrophobic property. Based on our preliminary
study, b-mangostin in its original form (non-formulated) showed strong
activity against Caco-2 cells (IC50= 8.27 μg/mL). It is expected that the
activity against multidrug-resistant cancer cells would be enhanced by
improving its formulation. The study was also designed to incorporate
b-mangostin to a PEGylated liposome loaded (Figure 22) with
doxorubicin to overcome multidrug-resistant of colon cancer cells to
provide a synergistic effect (Taher, 2021, unpublished).
(Mokhtari et al., 2017). The two most significant approval of the combination of drug nanocarrier
includes Vyxeos™ and chimeric antigen receptor T cells (CAR-T) therapy, Kymriah™ and or
Yescarta™ (Ghosh et al., 2019).
The b-mangostin is an active compound from G. mangostana with low solubility due to its
hydrophobic property. Based on our preliminary study, b-mangostin in its original form (non-
formulated) showed strong activity against Caco-2 cells (IC50= 8.27 µg/mL). It is expected that the
activity against multidrug-resistant cancer cells would be enhanced by improving its formulation. The
study was also designed to incorporate b-mangostin to a PEGylated liposome loaded (Figure 22) with
doxorubicin to overcome multidrug-resistant of colon cancer cells to provide a synergistic effect
(Taher, 2021, unpublished).
Drug Discovery and Development: Prospects and Challenges / 27
Figure 22: PEGylated liposome formulation with b-mangostin to improve
Figure 22: PEGylated liposome formulation with b-mangostin to improve delivery in colon cancer
delivery in colon cancer cells
cells
Brothers and sisters,
Ladies and Gentlemen,
Brothers and sisters,
Ladies and Gentlemen,
The Miracle Plant, Centella asiatica
THE MIRACLE PLANT, Centella asiatica
Introduction of C. asiatica
C. asiatica (L.) is an Asian native wetland medicinal herb commonly
Introduction of C. asiatica
known as pegaga, pegagan, daun kaki kuda, gotu kola, or Asian
C. asiatica (L.) is an Asian native wetland medicinal herb commonly known as pegaga,
pennywort. Besides its role as a medicinal plant for thousands of years,
pegagan, daun kaki kuda, gotu kola, or Asian pennywort. Besides its role as a medicinal plant for
particularly in the Ayurvedic system in India and as a traditional folk
medicine in China and Madagascar, C. asiatica has caught our attention
thousands of years, particularly in the Ayurvedic system in India and as a traditional folk medicine in
to study its unique properties. The herb possesses active principles
comprising triterpenoids, such as asiaticoside, madecassoside, and
asiatic acid (Figure 23). The huge attention on this plant is related to its 32
activity on skin generation, boosting memory, increasing concentration
and alertness, and anti-stress. During the wound healing process, C.
asiatica stimulates collagen synthesis, which allows the skin to proliferate
effectively.
China and Madagascar, C. asiatica has caught our attention to study its unique properties. The herb
possesses active principles comprising triterpenoids, such as asiaticoside, madecassoside, and asiatic
acid (Figure 23). The huge attention on this plant is related to its activity on skin generation, boosting
memory, increasing concentration and alertness, and anti-stress. During the wound healing process, C.
asiatica stimulates collagen synthesis, which allows the skin to proliferate effectively.
28 / Drug Discovery and Development: Prospects and Challenges
R1 R2 R3
Asiatic acid -H -H -CH3
Madecassic acid -OH -H -CH3
Asiaticoside -H 1)-β-D-glc-(6-1)- β-D-glc-( 4-1)- α-L-rha -CH3
Asiaticoside A1 -OH 1)-β -D-glc-(6-1)- β-D-glc-(4-1)- α-L-rha -CH3
Terminolic acid -OH -H -CH3
Asiaticoside B -OH 1)-β -D-glc-(6-1)- β-D-glc-(4-1)-α-L-rha -H
Asiatic acid Madecassic acid
Figure 23: The triterpenoids constituents of C. asiatica
33
C. asiatica has also been used by “traditional healers” (bomoh,
dukun, pawang) and aboriginal people in the southeast Asia region to
treat skin disease, measles, tonsilitis, and blood circulation problems
(Ahmad, 2002; Nor Hartina, 2017). Its popularity, however, is limited
Drug Discovery and Development: Prospects and Challenges / 29
to that of a vegetable rather than a therapeutic plant (Jabatan Perhutanan
Semenanjung Malaysia, n.d.). Varieties of C. asiatica that can be found
in Malaysia include pegaga cina or pegaga nyonya, pegaga daun lebar,
pegaga salad, and pegaga renek.
Brothers and sisters,
Ladies and Gentlemen,
Wound Healing Process
Damage to the integrity of biological tissue, such as skin, mucous
membranes, and organ tissues, is classified as a wound (Kujath &
Michelsen, 2008). Currently, millions of people are suffering from
chronic wounds worldwide. Chronic lower extremity ulcers do not heal
quickly and have become a serious concern for healthcare systems around
the world. Patients with wounds are susceptible to wound infection and
sepsis. In the United States, an estimated 6.5 million patients suffer from
chronic wounds, costing the healthcare system more than USD 25 billion
per year to address wound-related treatment (Järbrink et al., 2016). The
wound healing process involves a well-defined biological technique that
regenerates tissues. It entails a complicated series of events organised
into three distinct but overlapping phases: inflammation, proliferation,
and maturation (Kamoun et al., 2017).
The wound healing method involves complicated interactions
between different cell types, extracellular matrix components, and
cytokine mediators (Kamoun et al., 2017). Even though the natural
wound healing process begins to mend damaged tissues when a wound
is introduced, the wounds should be dressed appropriately. The dressing
should be able to intervene at the precise stage of wound healing or
provide the correct environment for the wound to improve the healing
process (Boateng et al., 2008; Kokabi et al., 2007).
A good and successful wound dressing should be able to maintain a
moist wound environment, protect the wound from subsequent infection,
heal the wound faster, prevent wound bed necrosis, and not cause further
trauma to the regenerated tissues after the dressing is removed. The
wound dressing should also be biocompatible with tissues and blood,
non-antigenic, non-toxic, and have adequate elasticity (Kamoun et al.,
30 / Drug Discovery and Development: Prospects and Challenges
2017). Autografts, allografts, and cultivated epithelial autografts, as well
as wound dressings made of biocompatible and biodegradable polymers,
are now used in wound healing.
Biocompatible polymeric hydrogels are considered the most
promising wound dressing materials, as they meet the effective wound
dressing requirements by providing an easy-to-handle dressing with no
irritation and no adherence properties, thereby maintaining or improving
the patients’ comfort (Boateng et al., 2008; Kokabi et al., 2007). A
medicated hydrogel dressing, which reduces the time for healing and
increases patient compliance, has now become very practical. The use
of hydrogel occlusive dressings to keep a moist wound environment has
proven to be an effective aid in wound healing. Asiaticoside hydrogel
is designed to hold moisture at the skin surface, offering a soothing and
gentle solution for dry, sloughy, necrotic wounds (eschar) as well as to
accelerate the wound healing process due to the presence of asiaticoside.
Currently, the available asiaticoside-based product is the Madecassol ,
®
which is applied as a wound healing cream.
Hydrogel
Hydrogels are hydrophilic, three-dimensional, water-insoluble polymeric
networks with high-sensitive to physiological environments and adequate
flexibility (Hamidi et al., 2008; Kamoun et al., 2017; Singh & Vashishtha,
2008). They can absorb a large amount of water, which makes them a
suitable material as a wound dressing for biomedical applications. Among
the various polymers that can be formed into hydrogel are Polyvinyl
Alcohol (PVA) and Polyethylene Glycol (PEG). PVA hydrogels are one
of the most well-known polymer gels with excellent biocompatibility
and attractive physical features, such as rubbery or elastic in nature, non-
toxicity, and non-carcinogenic properties (Gwon et al., 2010).
On the other hand, PEG is a water-soluble polymer that has sparked
a lot of interest for its potential applications in the biomedical field,
including wound healing, tissue scaffolding, and drug delivery. Due to
its non-toxic properties, PEG does not interfere with the immune system
and can be eliminated from the body quickly. PEG is also recognised for
its great chain flexibility and temperature resistance, which allows for the
Drug Discovery and Development: Prospects and Challenges / 31
formation of hydrogels with extremely high swelling capacity (Laine et
al., 2004). These properties make PEG practically perfect to be used as
the basis of in vivo hydrogel (Roberts et al., 2002).
The preparation of hydrogels usually involves the crosslinking
of polymer chains together to form a sheet or a film. To date, various
methods have been developed to prepare hydrogels, include chemical,
radiation, and freeze-thaw crosslinking (Yang et al., 2004). The freezing
and thawing crosslinking method avoids the toxicity and the leaching
problems with chemical crosslinking and producing a hydrogel with
better mechanical strength compared to the chemical and irradiative
techniques. Given that traditional herbal treatments have gained the focus
of scientific research in the last decade due to their safety feature and
economically viable technique of treating wounds and burns, hence, the
present study focused on developing a hydrogel formulation with wound
healing action containing the phytochemicals of interest. Among various
interesting plants is the C. asiatica Linn (gotu kola) or Asian pennywort.
Brothers and sisters,
Ladies and Gentlemen,
Development of Pegaga Research:
From Laboratory to Serum X
Hydrogel Formulation using C. asiatica
Three principal ingredients are found in C. asiatica comprising
asiaticoside, asiatic acid, and madecassic acid. All three components have
been identified to be clinically effective in the treatment of abnormal
scar formation, systemic scleroderma, and keloids (Hong et al., 2005).
Specifically, the asiaticoside exhibits a significant wound healing
activity (Shukla et al., 1999). It was reported that C. asiatica extracts
shortened the wound healing time significantly by acting more precisely
on the immediate process of healing (Poizot & Dumez, 1978). The most
valuable effect of the plant appears to be the healing of the scar through
the production of type 1 collagen and decreasing the inflammatory
response and myofibroblast production (Widgerow et al., 2000).
32 / Drug Discovery and Development: Prospects and Challenges
A proof-of-concept lab-scale study was carried out in the previous
research project (Malaysia Research Assessment (MyRA) incentives
grant no. MIRGS13010010006) with the main focus of fabricating
10 L of hydrogels based on the physical crosslinking of PVA by PEG
composite with asiaticoside to meet the requirement of enhanced wound
healing capability. Various properties and wound healing capability of the
hydrogels were investigated. The hydrogel containing asiaticoside was
poured into the gel cast (15 x 15 x 0.5 cm) where each gel cast contains
approximately 100 mL of hydrogels mixture. Currently, the Ministry of
Higher Education (MOHE) has allocated RM 103,000 to our research
team through the Prototype Research Grant Scheme (PRGS). The study
focused on the upscale production of hydrogel sheets with C. asiatica
for wound dressing application.
Pegaga Extraction
C. asiatica was extracted using the soxhlet extraction technique. The
whole part of C. asiatica was first weighed using a weighing balance
and washed before being dried in a laboratory dryer. The dried plant
was then grounded to obtain fine C. asiatica powder before the soxhlet
extraction system was applied using methanol. The combined crude
extract solutions were filtered using a filter paper and evaporated using a
rotary evaporator to obtain the extract in powder form (Figure 24) (Azis
et al., 2017; Ahmed et al., 2019).
The non-conventional extraction method was also performed using
the supercritical fluid extraction, which was screened using the two-level
factorial design and optimised through the Central Composite Design
(CCD) method by altering three factors (co-solvent percentage, pressure,
and temperature) on the asiaticoside content. The model obtained was
used to optimise the extraction process C. asiatica to obtain a higher
yield of asiaticoside fraction (Ruslan et al., 2020).
Optimisation of Fractionation
Two fractionation methods were performed for the optimisation of
pegaga extract, which is the fractionation using activated charcoal and
fractionation using silica gel. In the first fractionation, activated charcoal
and evaporated using a rotary evaporator to obtain the extract in powder form (Figure 24) (Azis et al.,
2017; Ahmed et al., 2019).
The non-conventional extraction method was also performed using the supercritical fluid
extraction, which was screened using the two-level factorial design and optimised through the Central
Composite Design (CCD) method by altering three factors (co-solvent percentage, pressure, and
temperature) on the asiaticoside content. The model obtained was used to optimise the extraction
Drug Discovery and Development: Prospects and Challenges / 33
process C. asiatica to obtain a higher yield of asiaticoside fraction (Ruslan et al., 2020).
Figure 24: Sample preparation, extraction and analysis of C. asiatica
Figure 24: Sample preparation, extraction and analysis of C. asiatica
Optimisation of Fractionation
was used according to a recent study with slight modification (Ruslan
Two fractionation methods were performed for the optimisation of pegaga extract, which is the
et al., 2020). The Vacuum Liquid Chromatography (VLC) technique
fractionation using activated charcoal and fractionation using silica gel. In the first fractionation,
was carried out using 75 g of activated charcoal mixed with 300 mL
activated charcoal was used according to a recent study with slight modification (Ruslan et al., 2020).
of distilled water and poured into the sintered glass funnel. The extract
The Vacuum Liquid Chromatography (VLC) technique was carried out using 75 g of activated
solution cannot pass through when the column was filled with dried
charcoal mixed with 300 mL of distilled water and poured into the sintered glass funnel. The extract
activated charcoal. Therefore, the activated charcoal containing water
solution cannot pass through when the column was filled with dried activated charcoal. Therefore, the
was vacuumed to remove the water. Approximately 50 g of C. asiatica
extract was diluted with 50 mL methanol (MeOH) and the aqueous extract
37
was poured onto the activated charcoal. The extract was then eluted with
100% MeOH and collected in a filter flask. Finally, a solution of methanol
and dichloromethane (a ratio of 3:2) was used to elute the extract and
the yield was collected before it was concentrated in a rotary evaporator.
For the silica gel fractionation method, 50 g of C. asiatica extract
was diluted with 50 mL of MeOH. The VLC was applied using 170 g
of silica gel (230–400 mesh ASTM) eluted in three different solvents,
which consist of 100% ethyl acetate (EtOAc), EtOAc: MeOH (1:1), and
100% MeOH. Four fractions of C. asiatica were examined on a silica gel
34 / Drug Discovery and Development: Prospects and Challenges
plate using the TLC to identify the presence of asiaticoside. All fractions
contained asiaticoside except the EtOAc fraction. The TLC profile spots
showed that the Rf value of the asiaticoside in the methanol:chloroform
mobile phase was 0.77, which was similar to the Rf value of the standard
asiaticoside.
HPLC Analysis
The HPLC technique was performed to determine the concentration
of asiaticoside content in each extracted sample. Based on the HPLC
analysis, the concentration of Activated Charcoal Fraction (CA
FAC), Silica Gel Ethyl Acetate Fraction (SGE FAC), Silica Gel Ethyl
Acetate:Methanol Fraction (SGEM FAC), and Silica Gel Methanol
Fraction (SGM FAC) were 10 mg/mL each, while the concentration of
asiaticoside standard was 1 mg/mL. All fractions and standards were
soluble in a methanol-water mixture. The retention time and peak area of
each sample were tabulated in Table 2. The CA FAC displayed the highest
peak area compared to other fractions with the peak value of 412 870 at
21.855 retention time. Therefore, CA FAC was selected for the upscale
production of the hydrogel. Figure 25 shows the HPLC chromatogram
of all fractions including the asiaticoside standard.
Table 2: The retention time and peak area for all samples including
the asiaticoside standard
Retention Peak
Samples
time value
Asiaticoside standard 21.789 832 861
Activated charcoal fraction (CA FAC) 21.855 412 870
Silica gel ethyl acetate fraction (SGE FAC) 22.078 28 888
Silica gel ethyl acetate: methanol fraction 21.144 48 526
(SGEM FAC)
Silica gel methanol fraction (SGM FAC) 21.971 248 906
Drug Discovery and Development: Prospects and Challenges / 35
Figure 25: HPLC chromatogram of (A) asiaticoside standard,
Figure 25: HPLC chromatogram of (A) asiaticoside standard, (B) C. asiatica sample fraction using
(B) C. asiatica sample fraction using Activated Charcoal (CA FAC),
Activated Charcoal (CA FAC), (C) Ethyl Acetate:Methanol Fraction (SGEM FAC), and (D)
(C) Ethyl Acetate:Methanol Fraction (SGEM FAC),
Methanol Fraction (SGM FAC)
and (D) Methanol Fraction (SGM FAC)
Brothers and sisters,
Brothers and sisters,
Ladies and Gentlemen,
Ladies and Gentlemen,
Upscale Production of Pegaga Hydrogel
Upscale Production of Pegaga Hydrogel
The CA FAC was selected for the upscale hydrogel production based on the TLC and HPLC
The CA FAC was selected for the upscale hydrogel production based
results. The hydrogel films were prepared under three different concentrations comprising 0.25%,
on the TLC and HPLC results. The hydrogel films were prepared under
0.5%, and 1.0% sample fraction using the freeze-thaw technique with the combination of PVA and
three different concentrations comprising 0.25%, 0.5%, and 1.0%
PEG as the basis of the formulation. After undergoing five consecutive cycles of 16 hours freezing at
sample fraction using the freeze-thaw technique with the combination
-80 °C and 8 hours of thawing at room temperature, the hydrogels exhibited a non-sticky and opaque
of PVA and PEG as the basis of the formulation. After undergoing five
appearance (Figure 26).
consecutive cycles of 16 hours freezing at -80 °C and 8 hours of thawing
at room temperature, the hydrogels exhibited a non-sticky and opaque
appearance (Figure 26).
39
36 / Drug Discovery and Development: Prospects and Challenges
Figure 26: Hydrogel samples with different concentrations after undergoing five consecutive cycles;
(A) control (without sample), (B) 0.25% of C. asiatica sample fraction, (C) 0.5% of C. asiatica
Figure 26: Hydrogel samples with different concentrations after undergoing
sample fraction, and (D) 1.0% of C. asiatica sample fraction
five consecutive cycles; (A) control (without sample), (B) 0.25% of
Figure 26: Hydrogel samples with different concentrations after undergoing five consecutive cycles;
C. asiatica sample fraction, (C) 0.5% of C. asiatica sample fraction,
(A) control (without sample), (B) 0.25% of C. asiatica sample fraction, (C) 0.5% of C. asiatica
and (D) 1.0% of C. asiatica sample fraction
sample fraction, and (D) 1.0% of C. asiatica sample fraction
Pre-Commercialisation of Pegaga Serum
Pre-Commercialisation of Pegaga Serum
Regarding its health benefit, especially, for healthy skin, the research on pegaga has extended
Pre-Commercialisation of Pegaga Serum
Regarding its health benefit, especially, for healthy skin, the research on pegaga has extended
Regarding its health benefit, especially, for healthy skin, the research
for the development of pegaga-based cosmetic products. The effectiveness of C.
for the development of pegaga-based cosmetic products. The effectiveness of C. asiatica has been asiatica has been
on pegaga has extended for the development of pegaga-based cosmetic
formulated as a serum preparation, namely Serum X (Figure 27), which was sponsored by Malaysia
products. The effectiveness of C. asiatica has been formulated as a
formulated as a serum preparation, namely Serum X (Figure 27), which was sponsored by Malaysia
Technology Development Cooperation (MTDC) under the commercialisation of R&D Fund (CRDF)
serum preparation, namely Serum X (Figure 27), which was sponsored
Technology Development Cooperation (MTDC) under the commercialisation of R&D Fund (CRDF)
grant scheme with financial aid totalling RM 69,000 for marketing purposes. The product is currently
by Malaysia Technology Development Cooperation (MTDC) under the
under market validation by one of the cosmetic companies for further development.
commercialisation of R&D Fund (CRDF) grant scheme with financial
grant scheme with financial aid totalling RM 69,000 for marketing purposes. The product is currently
aid totalling RM 69,000 for marketing purposes. The product is currently
under market validation by one of the cosmetic companies for further development.
under market validation by one of the cosmetic companies for further
development.
Figure 27: Serum X on the shelf during market validation
40
Figure 27: Serum X on the shelf during market validation
Figure 27: Serum X on the shelf during market validation
40
Drug Discovery and Development: Prospects and Challenges / 37
Output from Pegaga Research
Output from Pegaga Research
Our interest in pegaga research has produced some output, including:
Our interest in pegaga research has produced some output, including:
1. Research grants (3 research grants).
1. Research grants (3 research grants).
2. Human capital development (1 PhD, 1 Master, 7 Undergraduate,
2. Human capital development (1 PhD, 1 Master, 7 Undergraduate, 1 Research officer).
1 Research officer).
3. Article publication (3 indexes journal).
3. Article publication (3 indexes journal).
4. Patent (1 patent).
4. Patent (1 patent).
5. Book (1 book) (Figure 28).
5. Book (1 book) (Figure 28).
Figure 28: Book “Pegaga” available at Shopee.com.my
Figure 28: Book “Pegaga” available at Shopee.com.my
Brothers and sisters,
Brothers and sisters,
Ladies and Gentlemen,
Ladies and Gentlemen,
THE COVID-19 PANDEMIC The COVID-19 Pandemic
COVID-19 is caused by a novel coronavirus called Severe Acute Respiratory Syndrome
Coronavirus 2 (SARS-CoV-2 virus; formerly called 2019-nCoV) (CDC, 2021). The first case of this
COVID-19 is caused by a novel coronavirus called Severe Acute
respiratory-related illness was reported in Wuhan City, Hubei Province, China. After it was initially
Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 virus; formerly
reported to the WHO on December 31, 2019, the WHO declared the COVID-19 outbreak as a global
called 2019-nCoV) (CDC, 2021). The first case of this respiratory-related
illness was reported in Wuhan City, Hubei Province, China.After it was
initially reported to the WHO on December 31, 2019, the WHO declared
41
38 / Drug Discovery and Development: Prospects and Challenges
the COVID-19 outbreak as a global health emergency on January 30,
2020.As more cases were reported across different continents, the WHO
declared COVID-19 a global pandemic on March 11, 2020 (Gallegos,
2020).
At present, there are no specific treatments for COVID-19 infection.
The current treatment focused on alleviating the symptoms and providing
respiratory support, including mechanical ventilation, oxygen therapy,
hemodynamic support, and drug intervention using anti-microbial,
anti-viral, or other types of drugs. While mass vaccination is the most
effective approach to curb the spreading of the virus, other methods of
preventative measures and alternative medicine have been recommended
to treat COVID-19 as a short-term strategy.
The great Persian scholar Ibnu Sina, or commonly known in the West
as Avicenna (980–1037 AD) once recommended several preventative
strategies to implement during an epidemic outbreak, including
restricted movement control, self-isolation, fumigating the surrounding
environment with perfumed herbs (such as Ood, Kafoor, Sumbuluttib,
Saad Kofi, and Loban), and the use of appropriate antidotes (Tiryaqe
Wabai) and vinegar (Sirka) as prophylaxis. The anti-viral herbal approach
is based on single-use of Ziziphus jujuba, Cordia myxa, Cydonia oblonga,
Althea officinalis, Malva sylvestris, Crocus sativus, Aloe barbedensis,
Commiphora myrrha, Cinnamomum zeylanicum, which may be effective
for the prophylaxis of COVID-19 (Fatima et al., 2020)through the air,
water, utensils, fomite and feco-oral route blood. The pathogenesis and
clinical features of COVID-19 be the same as the clinical manifestation
associated epidemic Fever. In Unani medicine, various herbal drugs are
described under the caption of epidemic disease. Great Unani scholar
also Avicenna (980–1037 AD.
The pharmacological factor is necessary to treat the symptoms. In a
recent review, we reported the potential use of several drug intervention
that has been proposed as therapeutic agents in COVID-19 management,
such as Remdesivir, ivermectin, favipiravir, hydroxychloroquine,
dexamethasone, methylprednisolone, and CP (Capodice & Chubak, 2021;
Taher, et al., 2021).
Drug Discovery and Development: Prospects and Challenges / 39
Certain compounds have been tested to exhibit effective action
’ ’
against COVID-19 in silico, such as 5,7,3 ,4 -Tetrahydroxy-2’-
(3,3-dimethylallyl) isoflavone, myricitrin, methyl rosmarinate,
’ ’ ’
3,5,7,3 ,4 ,5 -hexahydroxy flavanone-3-O-β-D-glucopyranoside, and
’
(2S)-eriodictyol 7-O-(6 -O-galloyl)-β-D-glucopyranoside with a binding
affinity of -29.57, -22.13, -20.62, -19.10, and -19.47, respectively. The
target is the viral 3-chymotrypsin-like cysteine protease (3CL pro )
enzyme that controls the coronavirus replication and is essential for its
life cycle. The 3CL pro is a potential drug discovery target in Severe Acute
Respiratory Syndrome Coronavirus (SARS-CoV) as well as Middle East
Respiratory Syndrome Coronavirus (MERS-CoV) (Tahir et al., 2020).
In vitro and in vivo testing is also important to transform these potential
inhibitors into clinical drugs.
Realising the absence of an effective pharmacological intervention
to cure COVID-19 infection, achieving herd immunity from mass
vaccination becomes an effective strategy. Hence, the development of
vaccines is very crucial (Zhou et al., 2021). Given its proven record
in controlling infectious diseases, especially viruses (Yu et al., 2018),
governments and state agencies are encouraged to increase the coverage
of vaccination to reach vast herd immunity through vaccination as soon
as possible. As of June 25, 2021, there are 287 candidate vaccines with
103 of them under clinical evaluation worldwide and an additional 184
candidate vaccines under pre-clinical evaluation (WHO, 2021).
Over the past year, the world has been experiencing a tough and
challenging time during the COVID-19 pandemic. Efforts to establishing
new norms and the series of movement control orders across countries
and states have tremendously affected almost all daily activities and
sectors, including higher education, with difficulties to adapt changes
from face-to-face learning to online classes, from laboratory/clinical
activities to virtual mode and work-from-home orders.
The education sector should set a clear exit strategy on how to end
and break away from the COVID-19 nightmare. In view of this, IIUM
with its holistic education concept has been working hard towards
addressing the changes to stay active and relevant. According to the
40 / Drug Discovery and Development: Prospects and Challenges
Sejahtera Academic Framework (Borhan et al., 2021), IIUM has outlined
a transformation concept on humanising education for Rahmatan lil-
alamin. In the context of humanising education, IIUM introduced the
tagline ‘Khalifah, Amanah, Iqra’, Rahmatan lil-Alamin’ (KhAIR) to be
adopted as part of the graduate attributes. The KhAIR tagline is an Arabic
word, which translates for “good”. The KhAIR concept is a simplified
expression of the seven IIUM missions and it is hoped that the efforts
will contribute to the development of successful and well balanced IIUM
graduates post-COVID-19.
Brothers and sisters,
Ladies and Gentlemen,
Conclusion
Drug discovery and development are costly and timely. It takes 12–15
years and over USD 1 billion just to produce a single drug for a particular
disease. The failure rate is extremely high with only one would be
available in the market out of the thousands of drug candidates tested.
Despite its significant challenge to producing a safe and effective drug,
the process continues to achieve success thanks to the combined efforts
of the academic world and private industry. Driven by medical needs
and the emergence of new diseases, research on new drugs involves
the translation of basic scientific findings from a laboratory setting into
human use. Seyhan stated several reasons that impede the continuous
development of drugs, including poor hypothesis, irreproducible data,
ambiguous pre-clinical models, inappropriate statistical model, and
insufficient funding (Seyhan, 2019). These drawbacks may cause a
translational gap, which later he called as “valley of death” in drug
discovery and development. Concerning the development of C. asiatica
for medical use, it is still an on-going process, which requires funding
and support either from the government or industrial pharmacy for
further development. We also welcome other researchers to collaborate
on this project.
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