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Division of Pharmaceutical Biosciences
Faculty of Pharmacy
University of Helsinki
Finland
Evaluation of the Medicinal Uses and Antimicrobial Activity of Piper guineense
Schumach &Thonn
Eunice Ego Mgbeahuruike
ACADEMIC DISSERTATION
To be presented, with the permission of the Faculty of Pharmacy of the University of Helsinki,
for public examination in Auditorium 1041 at Biocenter 2 (Viikinkaari 5E) on June 7th 2019, at 12
o`clock.
Helsinki 2019
2
Supervisors Doctor Yvonne Holm, Ph.D.
Division of Pharmaceutical Biosciences
Faculty of Pharmacy
University of Helsinki, Finland
Professor Heikki Vuorela, Ph.D.
Division of Pharmaceutical Biosciences
Faculty of Pharmacy
University of Helsinki, Finland
Reviewers Doctor Heiko Rischer, Ph.D.
VTT Technical Research Centre Finland Ltd
Tietotie 2, FI-02044 VTT, Finland
Doctor Carina Tikkanen-Kaukanen, Ph.D.
Ruralia-Instituutti, Mikkeli
University of Helsinki, Finland
Opponent Professor Kemal Hüsnü Can Başer, Ph.D.
Department of Pharmacognosy, Faculty of Pharmacy
Near East University, Lefkoşa (Nicosia) N. Cyprus
© Eunice Ego Mgbeahuruike 2019
ISBN 978-951-51-5280-0 (paperback)
ISBN 978-951-51-5281-7 (PDF)
ISSN 2342-3161 (print)
ISSN 2342-317X (online)
Hansaprint, Helsinki, Finland 2019
3
TABLE OF CONTENTS
ACKNOWLEDGEMENTS……………………………………………………………………………...…….5
LIST OF ORIGINAL PUBLICATIONS ………………………………………………………...……………7
AUTHOR’S CONTRIBUTION TO THE ORIGINAL PUBLICATIONS………………………….….…..…8
LIST OF ABBREVIATIONS …………………………………………………………………….......……….9
ABSTRACTS…………………………………………………………………………………………….…...11
1. INTRODUCTION………………………………………………………………………….…….14
2. REVIEW OF THE LITERATURE…………………………………………………………...….16
2.1 Botanical description……………………………………………………………………………16
2.2 Traditional medicine in West Africa…………………………………………………………....17
2.3 Traditional and medicinal uses of Piper guineense ……………………………………….…....17
2.4 Antifungal activities of Piper guineense……………………………………………………………….18
2.5 Antibacterial activities of Piper guineense…………………………………………………..….19
2.6 Alkaloids and amides from Piper guineense…………………………………………………....20
3. AIMS OF THE STUDY……………………………………………………………….................21
4. MATERIALS AND METHODS………………………………………………………..……….22
4.1 Plant material……………………………………………………………………….…………...22
4.1.1 Extraction……………………………………………………………………………………..22
4.1.2 Chemicals, instruments and pure compounds………………………………………………...23
4.2 Antimicrobial screening………………………………………………………………………...23
4.2.1 Bacterial and fungal strains………………………………………………………………...…23
4.2.2 Agar disk diffusion method…………………………………………………………………...25
4.2.3 Microdilution turbidimetric broth method…………………………………………………….26
4
4.3 Method of analytical chemistry.............................................................................................…...27
4.3.1 Thin layer chromatographic conditions………………………………………………....…….27
4.3.2 HPLC-UV/DAD method………………………………………………………………….…..28
4.3.3 UHPLC/Q-TOF MS method………………………………………………………………….28
4.3.4 Method validation………………………………………………………………….………….28
4.3.5 Fractional inhibitory concentration (FIC) index …….………………………..………………29
4.4 Ethnobotanical survey………………………………………………………………….……….29
5. RESULTS AND DISCUSSION………………………………………………………………….33
5.1 HPLC and TLC methods development of P. guineense…………………………………...……33
5.1.1 Method application and validation.....................................................................................…...34
5.2 Ethnobotanical survey and field work………………………………………….……………….35
5.3 Antimicrobial activity…………………………………………………………….……………..38
5.3.1 Antibacterial activity………………………………………………………………………….38
5.3.2 Antifungal activity…………………………………………………………………………….42
5.4 UHPLC/Q-TOF MS results……………………………………………………….………….…44
5.5 Synergistic effects of piperine/piperlongumine and antimicrobials………………………….....45
6. CONCLUSION…………………………………………………………………………………..46
7. REFERENCES…………………………………………………………………………………...48
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ACKNOWLEDGEMENT
This thesis work was conducted at the Division of Pharmaceutical Biosciences, Faculty of
Pharmacy, University of Helsinki. I would like to thank the Head of the Division, Professor Heikki
Vuorela for accepting my proposal to start the doctoral program in the division. My sincere
gratitude also goes to Doctor Teijo Yrjönen who encouraged me to start the doctoral program by
first admitting me into the Faculty. I thank Professor Heikki and Teijo immensely for providing me
the opportunity and enabling environment to conduct a research in Pharmacognosy. This work was
made possible by the financial support from the University of Helsinki and from my family in
Nigeria. I would like to acknowledge the financial support inform of travel grant and the
dissertation completion grant awarded to me by the University of Helsinki and also from Ekhaga
Foundation Sweden.
I am very grateful to my major supervisor Professor Yvonne Holm for her thorough supervision,
correction and guidance which have paved way for the successful completion of my doctoral
studies. Yvonne is an excellent supervisor who always proffered solution to problems encountered
during the cause of my doctoral studies.
In a special way, I would like to thank Doctor Pia Fyhrquist for her collaboration and guidance in
the laboratory during the antimicrobial experiments. I thank Pia for her support and expertise in the
study of African medicinal plants as sources for antimicrobial compounds. I would like to thank
Professor Riitta Julkunen-Tiitto in whose laboratory we performed the HPLC-DAD and UHPLC-
QTOF-MS experiments.
I am very grateful to Professor Abraham Ngwuta and Mrs Chinyere Amandikwa of the Federal
University of Technology, Owerri Nigeria for their support and collaboration during the
ethnobotanical survey in Nigeria. I would like to acknowledge and thank Docent Into Laakso for the
technical assistance and scientific advice in the laboratory.
I would like to thank Doctor Festus Anasonye for offering valuable academic advice and for the
preliminary proofreading of the thesis book.
I am sincerely grateful to my colleagues in the Division of Pharmaceutical Biosciences, Faculty of
Pharmacy; Ilkka Miettinen, Elina Karhu, Karmen Kapp, Enass Salih, Maarit Kortesoja, Anna
Hiltunen, Eveliina Taavitsainen, Shella Gilbert-Girard, Inés Reigada, Krista Virtanen, Paola San
Martin Gaundo, Kirsi Savijoki, Malena Skogman, Docent Adyary Fallarero, and Docent Leena
Hanski, I appreciate the laugh and joke that we shared during personal interactions and seminars.
6
I would like to thank my Imo state and Igbo families in Helsinki for their words of encouragement
and support during the study. I really appreciate your numerous phone calls.
I will like to thank my family, my lovely mother, my husband Barrister Chijioke, my daughter and
my siblings for their love, care and support throughout this whole journey. I sincerely appreciate
you all.
Eunice Ego Mgbeahuruike
Helsinki, April 2019
7
LIST OF ORIGINAL PUBLICATIONS
The doctoral thesis is based on the following publications
I. Mgbeahuruike E.E, Yrjönen T, Vuorela H, and Holm Y. (2018). Optimization of thin-
layer chromatography and high-performance liquid chromatographic method for Piper
guineense extracts. Natural Product Communications, 13 (1), 25-28.
II. Mgbeahuruike E.E, Fyhrquist P, Julkunen-Tiitto R, Vuorela H, and Holm Y. (2018).
Alkaloid-rich crude extracts, fractions and piperamide alkaloids of Piper guineense possess
promising antibacterial effects. Antibiotics, 7(4), 98.
III. Mgbeahuruike E.E, Fyhrquist P, Julkunen-Tiitto R, Vuorela H, Amandikwa C and Holm
Y. (2019). An ethnobotanical survey and antifungal activity of Piper guineense used for the
treatment of fungal infections in West-African traditional medicine. Journal of
Ethnoparmacology, 229, 157-166.
IV. Mgbeahuruike E.E, Stålnacke M, Vuorela H, and Holm Y. (2019). Antimicrobial and
synergistic effects of commercial piperine and piperlongumine in combination with
conventional antimicrobials Antibiotics, 8(2), 55.
The publications were reprinted with the permission of the copyright holders and are referred to in
the text by their roman numerals.
Published article (review article) not included in the dissertation.
Mgbeahuruike E.E, Yrjönen T, Vuorela H, and Holm Y. (2017). Bioactive compounds from
medicinal plants: focus on Piper species. South African Journal of Botany, 112, 54-69.
8
AUTHOR’S CONTRIBUTION TO THE ORIGINAL PUBLICATIONS
I. The author collected the plant materials, participated in planning the experiment with the
co-authors, performed the extraction, performed the thin-layer chromatography
experiments, participated in the high-performance liquid chromatographic experiments,
participated in analyzing the data and interpreting the results, the author wrote the article
with the co-authors.
II. The author planned the experiment in collaboration with the co-authors, prepared the
plant materials and performed the sequential extraction, performed the antimicrobial
activity experiments, participated in performing the HPLC-DAD and UHPLC-QTOF-
MS experiments, participated in analyzing the data and interpreting the results, the
author wrote the article with the co-authors.
III. The author planned the experiment, conducted the ethnobotanical survey in
collaboration with the co-authors, prepared the plant materials and performed the
sequential extraction, performed the antifungal screening experiments, analyzed and
interpreted the results with the co-authors and wrote the article.
IV. The author participated in planning the experiments with the co-authors, participated in
performing the antimicrobial activity and synergistic evaluation, analyzed and
interpreted the results with the co-authors and wrote the article.
9
ABBREVIATIONS
AI Activity Index
AIDS Acquired Immune Deficiency Syndrome
ATCC American Type Culture collection
CFU Colony Forming Units
CLSI Clinical and Laboratory Standards Institute
DAD Diode Array Detector
FICI Fractional Inhibitory Concentration Index
GC Growth Control
HIV Human Immune-deficiency Virus
HPLC High Performance Liquid Chromatography
IZ Inhibition Zone
LOD Limit of Detection
LOQ Limit of Quantitation
MBC Minimum Bactericidal Concentration
MDR Multidrug-Resistant
MeOH Methanol
MFC Minimum Fungicidal Concentration
MIC Minimum Inhibitory Concentration
MS Mass Spectrometry
NaCl Sodium Chloride
PS Selectivity Value
QTOF Quadrupole Time of Flight
10
RPM Revolutions per Minutes
RSD Relative Standard Deviation
S Slope
SD Standard Deviation
SEM Standard Error of Mean
sp. Species
ST Solvent Strength
TLC Thin Layer Chromatography
TMP Traditional Medical Practitioners
UHPLC Ultra-High Performance Liquid Chromatography
UV Ultraviolet
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ABSTRACT
Piper guineense is a medicinal plant that has wide application in African traditional medicine where
it is often used in the treatment of bacterial and fungal infections. It is an economic plant with
numerous health benefits which is also consumed regularly as a functional food. The fruits, leaves
and seeds are used as spices and flavouring agents in commercial food preparations in West Africa.
The extracts are also used for the treatment of various diseases ranging from diarrhea, intestinal
diseases, rheumatoid arthritis, bronchitis, cough, stomach ache, asthma to febrile convulsions, fever
and mental disorders. There is also recent interest on the biological and pharmacological properties
of its bioactive compounds such as piperine, the main alkaloid constituents of P. guineense which is
responsible for its pungent aroma. Based on these numerous ethnobotanical, traditional and
economic uses of this plant, it became interesting to evaluate the bioactive compounds present in
the extracts and to further screen the extracts against some selected human pathogenic bacterial and
fungal strains so as to ascertain the efficacy of the extracts and its compounds as potent antibacterial
and antifungal lead compounds. Microbial resistance to the currently available antibiotics is a global
problem that has resulted to a constant search for a new antimicrobial drug with strong efficacy and
low cost. There is need to screen the extracts and bioactive compounds from P. guineense for
possible lead compounds for antibacterial and antifungal drug discovery.
In this study, first, a method was developed for the chemical profiling, qualitative and quantitative
analysis of P. guineense extracts and a good mobile phase composition was developed for the high
performance liquid chromatography (HPLC) and thin layer chromatographic (TLC) analysis of the
extracts. The effect of the chamber type on the separation was also evaluated using unsaturated
horizontal chamber in sandwich configuration, horizontal chamber in non-sandwich configuration
and twin-trough vertical chamber. Furthermore, the in vitro antibacterial activity of the extracts
were evaluated using 8 pathogenic Gram-positive and Gram-negative bacterial strains. An
ethnobotanical survey was also conducted on the use of P. guineense extracts in the treatment of
fungal infections in West African traditional medicine. The study area was chosen to be Imo state,
South Eastern Nigeria were P. guineense is mostly used by traditional healers for the treatment of
fungal infections which is often common among those suffering from HIV and AIDS. The aim of
the survey was to document the various methods of preparations and administrations of these
extracts for the treatment of fungal diseases. From this ethnobotanical approach, the leaves and
fruits extracts of the plant was further tested against 5 fungal strains including Cryptococcus
neoformans which causes meningitis in immunocompromised individuals.
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HPLC and TLC methods were developed for the analysis of P. guineense extracts with emphasis on
the shortest analysis time and minimal solvent consumption, and the best mobile phase giving
favourable resolution of bands was found to be toluene: ethyl acetate (PS 6-4 corresponding to
60:40 % v/v). The result of the TLC analysis showed that the developing chamber conditions does
not affect the TLC separation efficacy in the analysis of P. guineense extracts. From the HPLC-
DAD and UHPLC/Q-TOF MS investigation, piperine, dihydropiperine, piperylin, dihydropiperylin
or piperlonguminine, dihydropiperlonguminine, wisanine, dihydrowisanine, and derivatives of
piperine, and piperidine were identified in the extract of P. guineense. From the ethnobotanical
survey, it was recorded that the leaves and the fruits extracts of the plant is the predominant plant
part used for the treatment of fungal diseases in West African traditional medicine. A total of
twenty traditional medical practitioners (TMP) and herb sellers explained their methods of
administration of P. guineense extracts for the treatment of fungal infections. According to these
results, the oral intake of the extracts in locally produced bamboo alcohol (Kai-kai) is the most
common method of administration of the extracts for effective treatment. For the dosage, the TMPs
explained that they use a small glass tumbler which measures about 100 mL, administered 3 to 4
times in a day. The TMPs explained that they sometimes send their patients to the government
hospitals if the symptoms persist as a result of their failed treatment in order to avoid the death of
their patients. From the results obtained from this study, it could be seen that P. guineense fruit and
leaf extracts have activity against human pathogenic bacterial and fungal strains including
multidrug-resistant pathogens such as Pseudomonas aeruginosa. The extracts were active against
the tested bacterial and fungal strains with minimum inhibitory concentration (MIC) values ranging
from 19 to 2500 µg/mL. Significant antifungal and antibacterial activity were observed with the
lowest MIC of 19 µg/mL against Sarcina sp. A low MIC value of 39µg/mL was recorded for a
methanol fruit extract against Enterobacter aerogenes, Candida albicans, Bacillus cereus, Candida
glabrata and Candida tropicalis. Ethanol and hexane fruit extracts were effective against the
growth of Candida albicans, Staphylococcus aureus, Proteus mirabilis and Candida glabrata,
respectively, with a MIC of 78 µg/mL. Piperlongumine and piperine were active against the
bacterial and fungal strains with MIC values ranging from 19 and 156 µg/mL. The water extracts
were not active against the tested bacterial and fungal strains. Also, an in vitro biological and
synergistic effects of piperine and piperlongumine were tested in combination with conventional
antimicrobials (rifampicin, tetracycline and itraconazole) at various ratios. From the results, both
piperine and piperlongumine showed synergistic effect against Staphylococcus aureus when
combined at various ratios with rifampicin. Synergistic interaction was also observed with piperine
13
in combination with tetracycline against Staphylococcus aureus while antagonistic interaction was
recorded for piperlongumine and tetracycline against Staphylococcus aureus.
The result of the present study demonstrates that P. guineense extracts contain antibacterial and
antifungal compounds that may be useful for the discovery of new antibiotics.
14
1. INTRODUCTION
Piper guineense Schumach &Thonn, is a medicinal plant growing in various parts of Africa and
other parts of the world. It is commonly called Ashanti pepper or African black pepper, and has
been shown to be one of the most valuable spices with numerous health benefits (Ene-Obong et al.,
2018). Economically, P. guineense is a spice which is used in flavouring local dishes in West Africa
and the fruits are sold in local markets as condiments. It is a medicinal plant which has been utilized
traditionally in the treatment of various ailments and infectious diseases such as rheumatoid
arthritis, diarrhea, bronchitis, cough, intestinal diseases, stomach ache and asthma (Konning et al.,
2004; Ogunniran, 2009; Gbekley et al., 2017). It is also used in the treatment of mental disorders,
febrile convulsions, fever and to enhance female fertility (Oyemitan et al., 2015). It is a rich source
of antioxidants and has been found effective in the management of liver diseases (Oyinloye et al.,
2017). The leaves, seeds and fruits of this plant is used to prepare postpartum soup for women after
child birth as it is believed that it helps in the contraction of the uterus and cleansing of the womb of
women of possible remaining placenta after child birth and also enhances breast milk production
(Uhegbu et al., 2015, Ene-Obong et al., 2018). It has been shown to have effect in white blood cell
and red blood cell counts, thereby increasing the hemoglobin level (Uhegbu et al., 2015).
Biological studies have revealed that P. guineense extracts possess antifungal and antibacterial
properties (Okeke et al., 2001; Konning et al., 2004; Tekwu et al., 2012). P. guineense extracts have
been reported to have antifungal efficacy against various fungal strains, and could be a lead to new
antifungal agents (Ngane et al., 2003). It was found to be effective against Mycobacterium
tuberculosis which is the causative agent of tuberculosis, a life-threatening disease that is common
in the developing countries (Tekwu et al., 2012). The essential oils and the extracts possess
anticonvulsant, hypothermic, sedative, muscle relaxant, antipsychotic and anticonvulsant properties
(Oyemitan et al., 2015). The extracts and essential oils also have antioxidant and antidiabetic
activities and can be used in the treatment of depression (Okon et al., 2013; Oboh et al., 2013;
Oyemitan et al., 2015).
P. guineense contains valuable bioactive compounds which could serve as therapeutic agents for
drug discovery (Parmer et al., 1997; Adesina et al., 2003; Scott et al., 2005; Mgbeahuruike et al.,
2017), and these bioactive compounds such as alkaloids and amides have been reported to be
present in various parts of the plant (Adesina et al., 2003; Scott et al, 2005). Apart from its
antibacterial and antifungal properties, P. guineense extracts and bioactive compounds have been
reported to exhibit anticancer and antitumor efficacy and could be a potential lead to the discovery
15
of a new anticancer drug (Iweala, 2015; Bezerra et al., 2005). It contains piperine, an alkaloid with
interesting pharmacological properties which has been reported to have antibacterial, antifungal and
anticancer properties, and are also capable of enhancing the effectiveness of antimicrobial and
chemotherapeutic drugs (Nageswari et al., 2018; Bezerra et al., 2005; 2006; 2008).
The aim of this study is to evaluate the medicinal uses of P. guineense and to screen the extracts and
commercial compounds against some human pathogenic fungal and bacterial strains in search of
antimicrobial lead compounds. P. guineense is of great importance in traditional medicine, it
therefore became necessarily to broaden the knowledge on the antibacterial and antifungal activity
of the extracts so as to ascertain their acclaimed uses in the treatment of bacterial and fungal
diseases in traditional medicine. There is need to have also a readily available HPLC and TLC
methods for qualitative and quantitative determination of these bioactive compounds for possible
drug candidates.
16
2. REVIEW OF THE LITERATURE
2.1 Botanical description
P. guineense is a perennial climbing herb that is found growing mostly in tropical regions such as
Africa and other tropical zones of the world (Oyemitan et al., 2015). The plant belong to the genus
Piper of the Piperaceae family. It is often cultivated for its fruits, leaves and roots, by local farmers
who earn a living by selling it to traditional healers and the populace. It is a herb which could grow
up to 20m tall, climbing by means of its adventitious roots (Besong et al., 2016). It has various parts
which are utilized for different medicinal purposes. It consists of black berry fruits, seeds, leaves,
rhizomes and flower buds (Besong et al., 2016). The flowers which produces the fruits are greenish
yellow, the fruits when riped are reddish brown and occur in clusters and then produces the seed.
The fruit is small and round in shape (Fig. 1A) and has a hot pungent taste when chewed. The fruits
are sold in markets as flavouring agents (Freiesleben et al., 2015). The leaves are green and oval in
shape (Fig. 1B) and are used to prepare soup and decoctions for the treatment of various ailments. It
is called Uziza (Igbo), Odusa (Ibibio/ Efik) or Iyere (Yoruba) in Nigeria, West Africa. It has other
common names such as Guinea pepper, Benin pepper, False cubeb and Ashanti pepper (Besong et
al., 2016). Piper has numerous species which have been reported to have varying chemical
constituents based on their diverse nature (Besong et al., 2016, Oyemitan et al., 2015). Species
found in the same geographical region could have variations in their chemical constituents. It is a
vegetable plant that is used to enhance the taste of food.
17
Fig. 1. Piper guineense fruits (A) and leaves (B)
2.2 Traditional medicine in West Africa
West Africa is a region that is richly endowed with medicinal plants which are used for the
treatment of infectious diseases (Oguntibeju, 2018). The population which are mostly poor income
earners uses medicinal plants such as Piper guineense and Xylopia aethiopica for the treatment of
various diseases. The region owing to its numerous medicinal plants is a good source of a diversity
of bioactive compounds which could be potential lead compounds for drug discovery
(Olorunnisola, et al., 2013). According to World Health organization (WHO), greater number of
people in the developing countries like in West Africa rely on medicinal plants for the treatment of
infectious diseases. The population often patronize traditional medical practitioners (TMP) and herb
sellers for their primary health care needs. These TMPs collects medicinal plants which are easily
accessible from the wild or from their farms to prepare decoctions and various herbal formulations
for the treatment of diseases. Medicinal plants growing in tropical rainforest zone are rich sources
of bioactive compounds and as such the ethnobotanical information of such plants are needed for an
effective use as an antimicrobial lead compounds. In West Africa, the importance of traditional
medicine cannot be overemphasized because of lack of modern facilities in the rural areas (Ode et
al., 2011).
2.3 Traditional and medicinal uses of Piper guineense
Piper guineense is useful in traditional medicine and its nutritive and medicinal potentials have
been outlined in various pharmaceutical studies (Obodozie et al., 2010; Uhegbu et al., 2015;
Ekundayo et al., 1988; Ene-Obong et al., 2018). The fruits, seeds and leaves are often prepared in
alcohol or as decoctions together with other herbal formulations, and used in the treatment of
various diseases (Besong et al., 2016; Freiesleben et al., 2015). Ethno-pharmacologically, the roots,
leaves, fruits and seeds of P. guineense are relevant herbal products in African traditional medicine,
most importantly in West Africa where it is administered to nursing mothers to stimulate breast
milk production and to aid the contraction of the uterus after child birth (Okigbo and Igwe, 2007).
The fruits and seed extracts from this plant have been reported to be an important ingredient in the
preparation of Niprisan herbal formulation used to treat sickle-cell anaemia (Freiesleben et al.,
2015; Obodozie et al., 2010). Decoctions from the fruits and seeds are used in the treatment of
venereal diseases, rheumatism, gastrointestinal diseases and respiratory diseases (Udoh, 1999). P.
18
guineense extracts are used in the treatment of mental disorder and fever, and have been found to
possess sedative and muscle relaxant properties (Oyemitan et al., 2015). Extracts from P. guineense
are used as aphrodisiac and it has been reported that Yaji soup, which is often eaten as an
aphrodisiac in most West African countries, is prepared from the fruits of P. guineense (Ibrahim et
al., 2010; Asase et al., 2012). Infusions and decoctions from the fruits of P. guineense are
administered orally to treat bronchitis, cough, and intestinal diseases (Nwozo et al., 2017). Several
studies have shown that extracts of P. guineense could lower lipid peroxidation thereby preventing
inflammation and oxidative damage and are also used in the treatment of dysentery (Ogunniran,
2009; Nwozo et al., 2017). The leaves, fruits and whole plant parts of P. guineense are used to
prepare herbal formulation for the treatment of asthma and its related symptoms (Gbekley et al.,
2017). A previous research conducted on the ethnomedicinal uses of African medicinal plants has
reported that the leave extracts P. guineense are used in the treatment of sexually transmitted
diseases (Ajibesin et al., 2011). The fruits and leaves of P. guineense are ground and soaked in
alcohol with other herbs to prepare concoctions used for the treatment of epilepsy, convulsion and
malaria (Abila et al., 1993; Umoh et al., 2013). Previous research has shown that P. guineense
extracts are useful in the treatment of fertility disorder (Mbongue et al., 2005) and have been used
to stimulate sexual behavior in an adult male rat (Kamtchouing et al., 2002). It is also used as a food
preservative and as fragrance in perfume and cosmetic industries (Nwozo et al., 2017).
2.4 Antifungal activities of P. guineense
The effect of P. guineense extracts on various fungal pathogens has been reported (Dada et al.,
2013; Dzoyem et al., 2014; Abiala et al., 2015). P. guineense is effective against pathogenic fungal
strains such as Fusarium oxysporum, Fusarium solani, Macrophomina phaseolina, Fusarium
verticillioides and Botryodiplodia theobromae which are responsible for rots in watermelon fruits
and other vegetable fruits (Abiala et al., 2015). P. guineense extracts are often used as botanical
fungicides by local farmers because of high cost associated with synthetic fungicides. Evaluation of
the antifungal potentials of P. guineense extracts along with other Cameroonian spices, revealed
that it is effective against Candida albicans, Candida parapsilosis, Candida tropicalis, Candida
krusei, Candida lusitaniae, Cryptococcus neoformans, Candida guilliermondii and Candida
glabrata (Dzoyem et al., 2014). However the cold water maceration and hot water decoctions of the
plant extracts do not have activity against Candida albicans (Okigbo and Igwe, 2007). The activity
of P. guineense on Candida albicans demonstrates that its extracts could be effectively utilized in
19
treatment of infections caused by opportunists Candida strains in antifungal therapy. The antifungal
efficacy of the fruit and leaf extracts of P. guineense on various Candida strains (Candida albicans,
Candida tropicalis, Candida parapsilosis, and Candida glabrata) supports the use of the plant in
the treatment of toilet infections and sexually transmitted diseases. P. guineense extracts were used
as a natural insecticide to control maize weevil, Sitophilus zeamais (Asawalam, 2006).
Furthermore, Dada et al., 2013, reported that ethanolic and hexane extracts of P. guineense
inhibited the growth of Aspergillus flavus and Aspergillus niger. The result shows that P. guineense
has interesting antifungal properties and can be used as a good therapeutic agent in the discovery of
a new antifungal drug. Research has shown that fractions, natural compounds and extracts from P.
guineense can be explored as antifungal agents in the prevention of skin infection (Ngane et al.,
2003). The efficacy of these fractions and extracts on panel of organisms such as Microsporum
gypseum, Trichophyton mentagrophytes, Trichophyton rubrum, Aspergillus flavus, Scopulariopsis
brevicaulis, and Cryptococcus neoformans shows that they can be effectively used to combat
diseases and infections caused by these filamentous fungi.
2.5 Antibacterial activities of P. guineense
Bacterial infections often result to death if not well treated, and P. guineense has been widely
reported to exhibit antibacterial properties (Okeke et al., 2001; Konning et al., 2004; Anyanwu and
Nwosu, 2014). Extracts and fractions from various parts of this plant have antibacterial activity
against Gram-positive and Gram-negative bacterial strains (Tekwu et al., 2012; Dada et al., 2013).
It has been observed that the extraction solvent and method of extraction play a role in the
inhibitory activity of the extracts on the bacteria, and previous research have shown that hexane,
methanol and ethanol extracts are more effective than the water fractions (Dada et al., 2013,
Konning et al., 2004). However, essential oils from the fruits of P. guineense did not have activity
against Escherichia coli, Salmonella typhi, Klebsiella sp., and Pseudomonas aeruginosa
(Olonisakin et al., 2006). P. guineense is effective against Mycobacterium tuberculosis which is a
threat to human life (Tekwu et al., 2012). The ethanol extracts of P. guineense has been reported to
be effective against Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Salmonella typhi,
Klebsiella pneumoniae, and Proteus vulgaris (Okeke et al., 2001). The antibacterial evaluation of
the plant extracts on Streptococcus faecalis did not show remarkable activity (Okigbo and Igwe,
2007). The antibacterial activities of P. guineense on Acinetobacter spp., Bacillus cereus,
Escherichia coli, Salmonella spp., Shigella dysenteriae, Staphylococcus aureus by Dada et al.,
20
2013, revealed that bioactive compounds from the plant are good antibacterial agent and could be a
lead to the discovery of a new antibacterial drug. Previous research has shown that ethanol extracts
of P. guineense has remarkable activity against E. coli (Anyanwu and Nwosu, 2014), and these
researchers observed that the extracts inhibited the growth of B. subtilis, E. coli, and S. aureus with
the least activity observed in P. aeruginosa.
2.6 Amides and alkaloids in P. guineense
Amides and alkaloids from P. guineense and other Piper species are called piperamides and these
piperamide compounds have gained recognition in recent years for their strong efficacy as antibacterial,
antifungal, anti-inflammatory, and antitumor agents in drug discovery (Adesina et al., 2003; Nageswari et
al., 2018; Bezerra et al., 2005; Scott et al, 2005). Piperine, a potent alkaloid which has been reported
to have antimicrobial activity is contained in substantive amount in P. guineense (Adesina et al.,
2003, Scott et al., 2005). Other piperamide alkaloids found in P. guineense include piperylin, 4, 5-
dihyropiperlonguminin, piperlonguminin , and 4, 5-dihydropiperine (Scott et al., 2005). The
chemical structure of the piperamide compounds found in P. guineense are found in publication
(II).
21
3. AIMS OF THE STUDY
The general aim was to study and have more knowledge on the efficacy of extracts and bioactive
compounds of P. guineense, a medicinal plant and a spice that is highly valued in Africa because of
its numerous ethno-medicinal uses. To optimize the HPLC and TLC methods for the isolation of the
bioactive compounds and to evaluate the antimicrobial activity of its extracts and pure compounds
as potent antimicrobial agent.
Specific aims of the thesis were:
1. To optimize a thin-layer chromatography (TLC) and a high performance liquid chromatographic
(HPLC) method for the chemical profiling, qualitative and quantitative analysis of P. guineense
extracts setting emphasis on achieving the best possible overall separation of the main components
of the extracts (for example piperine) while keeping the analysis time and solvent consumption to a
minimum (I).
2. To investigate the antibacterial activity of various extracts and fractions from P. guineense using
pathogenic organisms such as Gram positive (Sarcina sp., Staphylococcus aureus, Bacillis cereus,
and Proteus mirabilis) and Gram negative bacterial (Enterobacter aerogenes, Escherichia coli,
Pseudomonas aeruginosa, and Salmonella enterica) (II).
3. To conduct an ethnobotanical survey and to investigate the antifungal activity of various extracts
and fractions from P. guineense using various fungal strains (Candida albicans, Candida tropicalis,
Candida parapsilosis, Candida glabrata and Cryptococcus neoformans) (III).
4. To investigate the in vitro pharmacological interaction between piperine and piperlongumine
when used in combination at various ratios with some conventional antimicrobials (tetracycline,
rifampicin and itraconazole) against Pseudomonas aeruginosa, Staphylococcus aureus and Candida
albicans (IV).
22
4. MATERIALS AND METHODS
4.1 Plant material
The fruits and leaves of P. guineense used in this study were collected from a rural village in Imo
State, South Eastern Nigeria in 2012 and 2013 by Eunice Ego Mgbeahuruike and Mrs Amandikwa
chinyere of the Federal University of Technology, Owerri, Imo state, Nigeria. Voucher specimens
were prepared and the plant materials were authenticated at the Department of Crop Science of the
Federal University of Technology, Owerri, Nigeria by Professor Abraham Ngwuta. Voucher
specimens were deposited at the herbarium of the Department of Crop Science of the same university
with the specimen number FUTO/SAAT/NS/005A for the fruit and FUTO/SAAT/NS/005B for the
leaf.
4.1.1 Extraction
The plant materials were powdered with an electric grinder to obtain finely ground samples. For the
TLC and HPLC optimization experiments in publication (I), 1.0 g each of dried powdered fruits and
leaves were extracted twice by sonication with 25 mL of methanol for 10 min. The extracts were
filtered with Whatman filter paper (Whatman GE Healthcare, Chicago, IL, USA) into a flask of
known weight. For the antibacterial and antifungal experiments in publications (II and III),
sequential extraction was carried out using solvents of varying polarities, starting with the least
polar solvent. First, 40 g of the plant material was extracted with 300 mL of hexane, followed by
extraction with 300 mL of chloroform, then 300 mL of ethanol and the residue was finally extracted
and washed with 300 mL of methanol. For each extract, the filtrate was evaporated using a
Rotavapor (Heidolph VV2000) combined with a water bath at +40C. In all, the extracts were
lyophilized for two days to dry completely. Macerations and hot water decoctions were also
prepared from the plant samples since these preparations are used in traditional medicine.
Macerations were prepared by weighing 10 g of the fruits and leaf plant materials into Erlenmeyer
flasks. 100 mL of water was added and extraction was performed for 24 hours using a magnetic
stirrer. The mixture was centrifuged at 2000 rpm for 15 min (Eppendorf AG centrifuge 5810R,
Germany). For the decoctions, 10 g of the plant material was boiled with 100 mL of water and
allowed to cool. The mixture was centrifuged for 15 min at 2000 rpm (Eppendorf AG centrifuge
5810R, Germany). Both the macerations and decoctions were carefully filtered using filter paper
(Schleicher & Schuell, ∅=150 mm, Germany), and freeze dried for two days in a lyophilizer. Prior
to the agar diffusion test, the freeze dried extracts were reconstituted and re-dissolved in their
corresponding solvents or in MeOH to a final concentration of 50 mg/mL for the antibacterial and
23
antifungal screening according to the method of Anyanwu and Nwosu, (2014) and Salih et al.,
(2017).
4.1.2 Chemicals, instruments and pure compounds
The solvents used for the study were HPLC grades of acetonitrile purchased from Merck KGaA
(Darmstadt, Germany), n-hexane (Merck), chloroform (Merck), ethanol (Sigma-Aldrich Corp.
(Merck), toluene (Merck), ethyl acetate (Merck), acetone (Merck), cyclohexane (Sigma-Aldrich
Corp. (Merck), and methanol (Merck) St. Louis, MO, USA. Ultrapure water was obtained, using a
Milli-Q water system (Merck Millipore, Billerica, MA, USA). Analytical grade piperine and
piperlongumine standard (≥ 97.0% purity) were purchased from TCI Europe N.V. (Zwijndrecht,
Belgium). TLC aluminium sheets coated with 20 x 20 cm silica gel 60 F254 (Merck, Germany) and
10 cm × 10 cm glass silica gel 60 F254 HPTLC plates (Merck, Germany). Tetracycline hydrochloride
(Sigma-Aldrich, St. Louis MO, USA), rifampicin (Sigma-Aldrich, St. Louis MO, USA), were used
as standard antibiotics for antibacterial investigation, while amphotericin B and itraconazole (Sigma-
Aldrich, St. Louis MO, USA) were used as positive controls for the fungal strains. Sterile Petri dishes
(∅ =14 cm, VWR Finland) were used for the screening. Other materials used includes; sterile
serological pipet (Falcon, Becton Labware Europe), Isosensitest agar (OXOID, Thermo Fisher
Scientific), Saboraud agar (OXOID, Thermo Fisher Scientific), nutient agar (Difco, VWR Finland),
sodium chloride (NaCl), sterile glass tube, sterile inoculation loops, sterile cork borers, Mueller
Hinton agar (Becton Dickson, New Jersey, USA), 96 well plates (Thermo Fisher Scientific,
Denmark). The instruments used were as follows: Multiskan microplate spectrometer (Thermo Fisher
Scientific), rotary evaporator (Heidolph instruments, Schwabach, Germany), Varioskan plate reader
(Thermo Fisher Scientific), lyophilizer (Heto LyoPro 3000, Denmark), and florescent microscope
(Tokyo, Japan).
4.2 Antimicrobial screening
4.2.1 Bacterial and fungal strains
The bacterial and fungal strains were obtained from the Division of Pharmaceutical Biosciences,
Faculty of Pharmacy, University of Helsinki, Finland. Most of the bacterial and fungal strains used
for the screening were model pathogenic organisms that are of clinical importance. The
characteristics of the bacterial and fungal strains used in the study are shown in Table 1. In all, the
24
growth inhibitory activity of the extracts were investigated using three Gram-positive bacterial
strains (Sarcina sp., Staphylococcus aureus ATCC 25923, and Bacillis cereus ATCC 10987), five
Gram- negative bacterial (Enterobacter aerogenes ATCC13048, Escherichia coli ATCC 25922,
Pseudomonas aeruginosa ATCC 27853, Salmonella enterica ATCC 43845 and Proteus mirabilis
43071), and five fungal strains (Candida albicans ATCC 10231, Candida glabrata ATCC 2001,
Candida tropicalis ATCC 750, Candida parapsilosis ATCC 7330, and Cryptococcus neoformans
ATCC 10226).
Table 1 Characteristics of the bacterial and the fungal strains used in the study
Pathogen
Diseases
Symptoms
Sarcina sp.
Gastric perforation,
emphysematous gastritis and
gastric ulcer
Vomiting, nausea,
bloating-like syndrome
in animals
Staphylococcus
aureus
Pneumonia, scalded skin
syndrome, blood stream
infection,
Fever, difficulty in
breathing, abdominal
pain, vomiting, nausea
Bacillus cereus
Gastrointestinal diseases and
diarrheal syndromes
Diarrhea, vomiting,
nausea
Enterobacter
aerogenes
Hospital-acquired infections such
as urinary tract infections, lower
respiratory tract infections, skin
and soft tissue infections.
Skin rashes and fever
Escherichia coli
infectious diarrhoea, urinary tract
infections, meningitis
fever, diarrhea,
abdominal pain,
dysentery
Pseudomonas
aeruginosa
Life-threatening nosocomial
infections, blood stream
infections
Fever, body pain,
diarrhea
Salmonella
enterica
Intestinal tract infections and
stomach flu
Stomach pain, bloody
stool, fever, Headache,
diarrhea
Proteus mirabilis
Pneumonia
Chest pain, cough, fever
Fungal species
Candida albicans
Candidiasis, oral thrush
Fever and painful
urination, discharge,
white spots in the mouth,
body pain
Candida
glabrata
Mucosal candidiasis and invasive
yeast infections
Thick discharge with a
burning sensation,
itching
25
Candida
tropicalis
Oral thrush
White spots in the mouth,
general body pain
Candida
parapsilosis
Urinary tract infection, joint
infection and meningitis.
Fever, abdominal pain,
vomiting
Cryptococcus
neoformans
Cryptococcal meningitis
Headache, neck pain,
fever and nausea
4.2.2 Agar disk diffusion method
Agar disk diffusion method as described in publications (II and III) were used for the antibacterial
and antifungal screening. A total of twelve extracts from the fruits and leaf extracts of the plant
were tested against the bacterial and fungal strains. Each of the extracts were prepared to a final
concentration of 50 mg/mL (stock solution). Amphotericin-B, rifampicin, tetracycline and
itraconazole were used as positive controls. The antibiotics were dissolved in methanol to a final
concentration of 10 mg/mL used for the test. Twenty-five mL of sterile base agar (Antibiotic agar
No. 2, Difco, VWR Finland) was applied as a bottom layer into the sterile Petri dishes using a
sterile, serological pipet (Falcon, Becton Labware Europe) and allowed to solidify, thereafter
twenty-five mL of Saboraud agar (OXOID, Thermo Fisher Scientific) was applied as the top layer
for the fungal strains and Isosensit agar for the bacterial strains. The Petri dishes were all allowed to
solidify, and then stored in +4C. The screening started with inoculation of the fungal/bacterial
strains onto solid agar slants which were incubated for 24 h (bacterial strains) or 48 h (fungal
strains) at +37°C. The viable microbial cultures from the agar slants were used to prepare an
inoculum for the test. Bacterial/fungi from the agar slants were transferred into 2 mL of 0.9% (w/v)
sodium chloride (NaCl) solution in a sterile glass tube using a sterile inoculation loop. 1 mL of the
suspension was transferred into another sterile glass tube, and the absorbance was measured at 625
nm (UV–Visible Spectrophotometer, Pharmacia LKB-Biochrom 4060). The other 1 mL of the
suspension (sterile part) was diluted with the 0.9% NaCl solution so that the absorbance at 625 nm
becomes 0.1 (this suspension contains approximately 1.5 108 CFU /mL). 200 µL of this diluted
fungal/bacterial suspension was spread evenly on each Petri dish and left to dry for some seconds
with the lid open. A sterile cork borer (11mm diameter) was used to make six holes equidistantly
from each other on the agar surface of the Petri dishes. 200 µL of the 50 mg/mL plant extracts and
200 µL of the 10 mg/mL antibiotics were carefully pipetted into the holes respectively. Methanol,
ethanol, hexane and chloroform, 200µL of each, were used as solvent controls respectively. The
Petri dishes were incubated for 24 h (bacterial strains) or 48 h (fungal strains) at +37 °C. The
26
diameters of the zones of inhibition were measured with a caliper under a Petri dish magnifier and
expressed as the mean of the diameters of three replicates ± SEM.
The Activity index (AI) of the various extracts were measured in relation to the standard antibiotics
according to Fyhrquist et al., (2014).
4.2.3 Microdilution turbidimetric broth method
The microdilution turbidimetric broth method was used for MIC, MBC and MFC estimation as
described in publications (II, III and IV). This was estimated based on the guidelines of Clinical
and Laboratory Standards Institute (CLSI) (Cockerill et al., 2012). From the result obtained from
the agar disk diffusion assay, minimum inhibitory concentration (MIC) was estimated for some
selected extracts based on their good antibacterial/antifungal activity. MIC is considered to be the
lowest concentration of an extract or compound resulting in the inhibition of at least 90% of the
growth of a fungal strain. Only extracts which expressed marked antibacterial/antifungal activity in
the agar disk diffusion assay were tested for MIC. For the MIC evaluation, two-fold serial dilutions
of the extracts from 9.75-2500µg/mL were prepared in sterile Mueller Hinton broth (for bacterial
strains) and Saboraud broth (for fungal strains). Commercial pure compounds, piperine (1mg/mL
concentration in methanol) and piperlongumine (1mg/mL concentration in methanol) were also
two-fold serially diluted in Mueller Hinton/Saboraud broth. Itraconazole and amphotericin-B or
rifampicin and tetracycline were each two-fold serially diluted in Saboraud broth or Mueller Hinton
broth from 0.48-125 µg/mL respectively. The absorbance of 1 mL of the 48 h bacterial/fungal
cultures were measured for turbidity at 625 nm using a UV–Visible Spectrophotometer type 1510
(Thermo Fisher Scientific Oy). The absorbance was adjusted to 0.1 at 625 nm (approximately 1.0 ×
108 CFU/mL). 100µL of this suspension A625= 0.1 was further diluted 100-fold to get a working
suspension or inoculum containing 1.0 106 CFU/mL. 100µL of this inoculum, and 100 µl of the
plant extracts, pure compounds, antibiotics, and solvent controls, were pipetted into the 96 well
microtiter plates. Therefore, each well contained 5 × 105 CFU/mL. The solvent controls contained a
maximum of 5 % (v/v) of each solvent to be tested for toxicity. The growth control (GC wells)
contained only the bacterial/fungal suspension, and the test wells (T wells) contained plant extracts
or pure compounds + bacterial/fungal suspension. Moreover, sample controls wells were prepared
for each plant extract/compound to be tested, and these wells contained plant extract/pure
compound and the broth only. The microwell plates were incubated for 48 hours in an incubator
coupled to a shaker at +37C. The tests were done in triplicate and the % growth was expressed as
the mean of these triplicates standard error of mean (SEM). The minimum bactericidal
27
concentration (MBC) or minimum fungicidal concentration (MFC) was evaluated by pipetting
100µL from those wells of the microtiter plate, which contained 2 and 4 times higher concentrations
than their MIC values on Petri dishes ( = 9 cm) containing Isosensit/Saboraud agar, and
incubating the dishes for 24 h or 48 h at +37°C. The MBC or MFC was taken as the lowest
concentration where no visible growth on the Petri dish was observed after the incubation.
4.3 Methods of analytical chemistry
4.3.1 Thin-layer chromatographic conditions
100 mg of the samples were dissolved in 1 mL of methanol (stock solution), and a 5 mg/mL
concentration was prepared from the stock solution for the TLC analysis in publication (I). The
extract was applied on the plate using CAMAG linomat IV TLC spotter (CAMAG AG, Muttenz,
Switzerland).TLC aluminium sheets coated with 20 x 20 cm silica gel 60 F254 (Merck), cut to 4 cm ×
10 cm, were used for testing the various mobile-phase compositions. A CAMAG REPROSTAR 3
was used to view the plates at 254 nm. Aliquots of 5 µL of the sample solutions were applied on the
plate as 6 mm bands, using the Linomat IV application device (CAMAG). The application speed was
6 sec/µL, plate width 40 mm, band 6 mm, space 6 mm, starting position 10 mm and development
over a path of 8 cm. Solvents of different solvent strength (ST) were combined in various proportions
and tested under the same experimental conditions with 10 mL of the mobile phase. The solvents
tested include toluene, ethyl acetate, n-hexane, acetone, cyclohexane and methanol.
HPTLC precoated silica gel 60 F254 plates were used for evaluation of the effects of the developing
chamber on the separation. A CAMAG twin-trough vertical chamber and CAMAG horizontal
chamber were used. HPTLC was performed on 10 cm × 10 cm glass silica gel 60 F254 HPTLC
precoated plates (Merck). The plates were developed in an unsaturated 10 cm × 10 cm twin-trough
chamber to a distance of 8 cm. The plates were documented under ultraviolet (UV) radiation at 254
nm. The optimum mobile phase of toluene-ethyl acetate 6:4 v/v was used. Three different volumes
of 3, 4 and 5µL of the 5 mg/mL concentration of the fruit and leaf extracts were used. The procedure
included a plate width of 100 mm, band 8 mm, space 5 mm, starting position 15 mm, 6 sec/µL and
development over a path of 8 cm while 10 mL of the solvent were used for the twin-trough chamber
and 2.5 mL for the horizontal chambers.
4.3.2 HPLC-UV/DAD method
28
For publication (I), the samples were analyzed with an HPLC Waters Tm 717 autosampler,
equipped with a photodiode array detector set at 240 nm (Waters Corp., Milford, MA, USA). Two
initial experiments were conducted at different gradient times of 30 and 60 min, with a total run
time of 25 min each. The HPLC conditions were described in the original publication (I). The
simulation with DryLab software was done with DryLab 2010 version 3.1 (LC Resources Inc.,
Alamo, CA, USA; in Europe: Molnar, Berlin, Germany). The retention time and peak area from the
chromatographic data were input into the DryLab software data entry. Peak-matching functions and
UV were used to match the peak identities. Gradient runs were predicted with the DryLab software
simulation, and a resolution map was used to predict the optimum separation conditions.
For publication (II), the samples were analyzed with an HPLC Waters 600 E pump and a controller
equipped with a 991 photodiode array detector. Samples were injected using an autosampler
controlled by Agilent Chemstation software (Water Corp., Milford, USA). The HPLC conditions
were described in details in publication (II). The UVλ absorption maxima spectra of the major
components in the P. guineense extracts were recorded between 200 and 400 using Agilent
Chemstation software and the compounds in the extracts were identified by comparing the retention
times and UV spectra with that of commercial reference standard (piperine) and also with previous
literature (Adesina et al., 2003; Scott et al., 2005).
4.3.3 UHPLC/ Q-TOF MS method
The masses of piperamide alkaloids were identified using UHPLC-DAD (Model 1200 Agilent
Technologies)-JETSTREAM/QTOFMS (Model 6340 Agilent Technologies) equipped with a 2.1×
60 mm, 1.7 μm C18 column (Agilent technologies) equipped with a 2.1× 60 mm, 1.7 μm C18
column (Agilent technologies) according to Taulavuori et al., 2013. The gradient range was from 0
– 50% of solvents A (aqueous 1.5% tetrahydrofuran + 0.25% acetic acid) and solvent B (100 %
methanol) and flow rate 0.4 mL/min. Mass spectrometry in positive and negative ion mode,
depending on the compounds were used to get the (M+Na or H-) ions.
4.3.4 Method validation
For method validation the parameters were determined, using a piperine standard solution diluted to
appropriate concentrations. Parameters such as linearity, limit of detection (LOD), limit of
quantification (LOQ), precision (interday and intraday) and extraction recovery were evaluated to
ascertain the sensitivity and accuracy of the method. A stock solution of 1 mg/mL concentration of
piperine was prepared in methanol to get six working standard solutions ranging from 2 to 10 µg/mL
29
(six points) for the calibration curve. For the LOD and LOQ, six working standard solutions were
prepared, and a more serial dilution of the standard solution of piperine was carried out in decreasing
concentrations. The precision was determined by injecting six replicates of the standard solution on
the same day and on 3 consecutive days. The validation methods are described in detail in publication
(I)
4.3.5 Fractional inhibitory concentration (FIC) index and Isobologram (IV)
The fractional inhibitory concentration (FIC) index was used to estimate the synergy (Kang et al.,
2011; Abreu et al., 2017 ).This method supplies a graphic demonstration with linearly arranged x
and y axes which is plotted using FIC (A) and FIC (B). For the FIC index calculation, the MIC
values of five combination ratios each of piperamide/antimicrobial were used to calculate FIC (A)
and FIC (B) using the equation below:
The FIC values were calculated as follows.
𝐹𝐼𝐶(𝐴)=MIC value of combined piperamide and antimicrobial
MIC value of antimicrobial alone
𝐹𝐼𝐶(𝐵)=MIC value of combined piperamide and antimicrobial
MIC value of piperamide alone
𝐹𝐼𝐶 𝑖𝑛𝑑𝑒𝑥 = 𝐹𝐼𝐶(𝐴)+ 𝐹𝐼𝐶(𝐵)
A FIC index value of ≤0.5 was interpreted as synergy. The FIC index was calculated on the basis of
the above-mentioned equation where FIC index = X + Y and interactions defined as; FICI ≤ 0.5,
synergy; > 0.5–≤ 1.0, additive; >1.0–≤2.0, indifference; and >2.0, antagonism (Kang et al., 2011)
4.4 Ethnobotanical survey
The ethnobotanical survey was conducted in Imo state, where P. guineense is frequently used as
herbal remedy in the treatment of candidiasis and other fungal diseases (publication III). Imo state
is situated in South-Eastern Nigeria and share boundary with the South-South geo-political zone of
Nigeria. It is a tropical rain forest zone located between 4°45 -7°15N and 6°50 - 7°25E, covering a
30
total land area of 5,530 km2. The field work was conducted in ten localities between November and
December 2017. A total of 20 traditional healers and herb sellers were interviewed (two from each
locality). The purpose of the work was to validate the use of P. guineense in the treatment of fungal
diseases. In the survey, a house to house strategy was used with the permission of the village heads
and traditional rulers. The questionnaire used in the ethnobotanical survey is shown in figure 3. The
village heads were notified prior to the investigation and the traditional healers were also well
informed. The investigating team obtained an ethical approval from each of the village heads.
Detailed and validated questionnaires were administered to twenty traditional healers. The
questionnaires were written in English, but the local language (Igbo) was used during the interviews
and conversations.
Figure 2. Map of Nigeria (http://www.maparchive.org) showing the study area Imo state shaded in a
black square arrow.
31
Figure 3. Questionnaire for the ethnobotanical survey on the traditional use of Piper guineense for
the treatment of fungal infections in Imo state, South Eastern Nigeria.
1. Gender Male □ Female □
2. What is your name (optional)……………………………………………………………..
3. What is your major occupation? : a). Herbalist □ b). Traditional medical practitioner □
c). Traditional healer/ herb seller □ d). Other □
4. What is your age? : a). 20-35 □ 36 – 55 □ 56 – 75 □
5. For how long have you been practicing traditional medicine? : a) 1 – 15 years □
16 -30 years □ 31 years and above □
6. What is your source of knowledge of traditional medicine? : a). Inherited/learnt from parents □
Training from other herbalists □ Other □
7. What is your level of education? : a). University education □ High school □
Primary education □ No formal education □
8. What is your religion? : a) Christianity □ Traditionalist □ Islam □ Other □
9. Can you name some of the fungal diseases that you treat with Uziza (Piper guineense)?
10. How often do you treat these diseases?
11. What are the symptoms and how do you diagnose this fungal infections?
Fungal infections
Local name
Method of diagnosis/
symptoms
32
12. Which part of the plant do you often use?: Fruit □ Leaves □ Roots □ Other □
13. How do you get the Piper guineense used for the treatment? : a) Cultivate them □ b) Buy
from the market □ c) Collect from the wild forest and bushes □ d) Other □
14. What are the method of preparations and administrations?
Fungal diseases
Method of preparation
Mode of administration
15. What are the dosage of administration and how long does the treatment last?
16. What do you do if you observe that the patient is not responding to your antifungal treatment?
Do you have some general comment about the possible challenges you face in your daily treatment
of fungal infections using Piper guineense extracts?
33
5. Results and discussion
5.1 HPLC and TLC methods development of Piper guineense (I)
The HPLC experiment conducted with dryLab simulation program gave a good and favorable
resolution of the main components of the extracts. The aim which was to have the best possible
overall resolution while keeping the analysis time and solvent consumption to a minimum was
achieved in a minimum number of runs. With DryLab software, it was possible to develop an
optimum condition for the analysis of P. guineense extracts. The optimized experiment which
began with two initial runs of a 30 min and 60 min gradient was found to be a 19 min binary
gradient with the proportion of organic solvent increasing from 39% to 80.4%, flow rate of 1
mL/min and injection volume of 20μL. This method was achieved by inputting the data (dwell
volume, gradient conditions, column length, column diameter and flow rate) from the two initial
experimental runs into DryLab. The HPLC conditions for these two initial gradient runs are
described in details in publication (I) Equal volumes of the sample were injected during the two
initial experimental runs in order to maintain constant peak areas. Using the experimental data
obtained from these initial runs, an optimum gradient run was predicted. This was possible by the
use of resolution map, peak-tracking and peak matching functions which were used to make
adjustments so as to predict a new chromatogram with emphasis on analysis time, solvent
consumption and optimum resolution of the peaks. The simulated separation conditions were
experimentally confirmed and the simulated separation was very similar to the actual experimental
separation. The method achieved in the experiment was the best separation condition, with the
shortest run time and good peak shape for the analysis of Piper guineense extracts. Based on
previous literature and by the use of UV spectra of reference standards and their retention times, the
compounds were identified in the extracts. The UV spectra of the major components in the P.
guineense extracts were taken, based on the chromatogram of the optimized 19 min run. From the
result, it was observed that the overall resolution and the resolution of the critical peak pairs with
the optimized method was better than or equal to one previously published method (Scott et al.,
2005), albeit at the cost of a slightly longer analysis time. The HPLC experimental method used in
these analyses provided satisfactory overall resolution for most of the main piperamides of P.
guineense and the method was found to be simple, sensitive and accurate. The developed HPLC
method was applied to determine the percentage content of piperine in P. guineense which was
found to be 0.43 % w/w, linearity (0.997), interday precision (% relative standard deviation (RSD),
34
1.6), intraday precision (% RSD, 2.7 – 5.9), recovery (98.4%), limit of detection (0.001 μg /mL)
and limit of quantification (0.003 μg /mL).
In the TLC experiment, the mobile phase composition which was systematically tested using
various proportions of solvents differing in ST and PS values under the same experimental
conditions according to the method of (Nyiredy, 2002), gave a good separation of the main
components of the extracts. The group, ST and PS values of individually selected solvents were
described in details in publication (I). The TLC experiment was aimed at achieving the best
possible overall separation of the main components of the extracts and the mobile-phase
composition giving these favorable resolution of the bands was found to be toluene: ethyl acetate
(PS 6-4 corresponding to 60:40 % v/v). The selection of the solvent combinations tested during the
method development were based on previous literature on the TLC analyses of extracts of various
Piper species. Most of the solvents have been previously used in the TLC analysis of various Piper
species but not Piper guineense. From the review of literature, bicomponent solvent mixtures
containing ethyl acetate as the other solvent appeared to be the most widely used, also it was
observed that acetone had been applied for the task. The effects of the developing chamber which
was tested using three types of unsaturated chamber conditions: horizontal chamber in sandwich
configuration, horizontal chamber in non-sandwich configuration and twin-trough vertical chamber
showed that the developing chamber conditions does not affect the TLC separation efficacy in the
analysis of P. guineense extracts. The mobile phase giving the best resolution of the bands which
was found to be toluene-ethyl acetate (PS 6:4 v/v), was applied for this task. Different volumes of 3,
4 and 5µl of the fruit and leaf extracts were applied on HPTLC precoated silica gel 60 F254 plates
after which there was no observed effect in the separation efficacy of P. guineense extracts. The
result shows that the choice of chamber type should be made based on any other criteria during the
TLC analysis of P. guineense extracts.
5.1.1 Method application and validation (I)
In publication (I), the developed method was applied for the rapid estimation of piperine from P.
guineense. Piperine was found to constitute 0.43% w/w of the seed of P. guineense. Piperine is
important because of its diverse biological and therapeutic potentials in recent pharmacological
studies (Chonpathompikunlert et al., 2010). In the method validation, calibration curve was linear
over a range of concentrations and the linearity was favorable and fell within the concentration
35
range of 2–12 μg/mL (Fig. 4). The R2 with respect to peak area was 0.997 while the slope and
intercept were 75 267 and 354.84, respectively.
Fig. 4. Calibration curve of piperine showing the coefficient of determination (R2)
The results obtained from the LOD (0.001 μg/mL) and LOQ (0.003 μg/mL) revealed that the
method has favorable sensitivity. In the experiment, LOD was evaluated and calculated to be the
lowest concentration of piperine that could be detected. From the calibration curve, these
parameters were calculated as the relative standard deviation (RSD) of the response and slope (S).
The interday and intraday precisions evaluated during the experiment were within the range of 1.6 –
5.9%, thus, this range was satisfactory and acceptable and revealed that the method was
reproducible. The method can be used to analyze extracts and fractions produced from P.
guineense, and also can be used as a quality-control tool by pharmaceutical industries to explore
piperine and other active compounds in P. guineense for drug discovery and for other therapeutic
purposes.
5.2 Ethnobotanical survey and field study (III)
P. guineense is predominantly used in the treatment of fungal infections by traditional healers in
Imo state, South Eastern Nigeria and for other various ethno medicinal purposes (Besong et al.,
36
2016). The result obtained from the ethnobotanical survey shows that the leaves and fruits are the
mostly used plant parts for the treatments of fungal infections in Imo state. It was observed that the
oral intake of the extracts in locally produced bamboo alcohol (Kai-kai) is the most common
method of administration. The traditional healers explained the various methods of preparations and
administrations of the decoctions and concoctions from the roots, leaves, and fruits of P. guineense
for the treatment of infectious diseases. The 20 traditional healers (14 male and 6 female) were
between the ages of 40 to 70 years. There were more male than female traditional healers and most
of them have long been practicing traditional medicine. The findings from our study is in line with
previous findings that there are more male than female traditional healers in Africa (Cheikhyoussef
et al., 2011; Ngarivhume et al., 2015). Most of the traditional healers have practiced traditional
medicine for over 25 years as their only source of income. They explained that they have inherited
the practice from their parents. It was observed that few of the participants have university
education and have recorded the information on the preparation and administration of P. guineense
extracts on their various exercise books. The healers always obtain the plant materials for their
traditional healing practice from their farms or from the markets.
The traditional healers sometimes sell P. guineense fruits and leaves which they have harvested
from their farms in local markets as spices and medicine to the general population for the purpose
of making profit. This explains that the P. guineense extracts are readily available and that the
traditional healers do not have problems in getting the plant materials used for the herbal treatments
since they are sold in the local markets. The symptoms given by all the healers on the methods of
diagnosis of the fungal infections is similar, so also the method of preparations (hot infusion,
decoction in combination with Xylopia aethiopica and then the plant material is soaked in mild
alcohol). The figure below shows one of the traditional healers in his herbal clinic where he
displayed various herbal preparations from P. guineense and He is referred to as “Doctor” in his
community.
37
Fig.5. P. guineense herbal formulations displayed by a herb seller (left) in Egbu community, Owerri
North Local Government Area, Imo state, South-Eastern Nigeria.
Sometimes the healers send their patients to the government hospitals if the symptoms persist as a
result of their failed treatment or lack of proper understanding of the type of fungal infection to
treat. It was observed that oral intake is the most common method of administration and this
correlates with the findings of Maroyi, (2013) that herbal preparations are mostly administered
orally by traditional healers. According to the traditional healers, the decoctions made from the
fruits and leaves, prepared in mild alcohol is mostly effective when administered orally for the
treatment of fungal diseases expressed by thrush on the tongue or candida vaginosis. The dosage is
usually measured with a small glass tumbler which can be estimated to be about 100 mL, and it is
often taken 3 to 4 times in a day. Leaves and fruits are the most frequently used plant parts for the
treatments. This correlates with the findings of Rahmatullah et al., (2012), who have reported that
the leaves of medicinal plants are used more often in traditional medicine than the other plant parts
perhaps because leaves are easy to collect. The demographic characteristics of the respondents are
shown in figure 6. The ethnobotanical result indicates that P. guineense is an important medicinal
plant which is highly utilized in the treatment of fungal infections in Imo state, South- Eastern
Nigeria and could be a source for the discovery of a new antifungal scaffolds for drug discovery.
38
Fig 6. The demographic characteristics of the respondent shows the number of traditional healers
with regards to gender, occupation, age range, years of practice (experience), sources of knowledge
of traditional medicine, level of education and religion. The data figure shows the number of
respondents.
5.3 Antimicrobial activity
5.3.1 Antibacterial activity
P. guineense is an African medicinal plant used by traditional healers for various medicinal
purposes and more often, as herbal remedies for the treatment of symptoms related to bacterial
infections, such as diarrhea, cough and rashes, and thus these uses are now justified by this study.
Piper guineense extracts revealed potent antimicrobial efficacy against various Gram-positive and
Gram-negative bacteria (II). Among all the bacterial strains tested in this study, Enterobacter
aerogenes which is responsible for most hospital-acquired infections (Jha et al., 2016) was screened
for the first time in this study and a large zone of inhibition (49.7 mm) was observed with the
methanol extract of the fruit of the plant with MIC value of 39 µg/mL and MBC value of 78 µg/mL
respectively (publication II). This bacterium causes urinary tract infections and lower respiratory
tract infections. The other extracts of the fruit of P. guineense showed promising inhibitory activity
against Enterobacter aerogenes also. The antibacterial efficacy of the extracts correlated with
previous research results that plant-derived compounds and plant extracts could be potential sources
for new antibacterial drugs against multi-drug-resistant (MDR) pathogens (Subramani et al., 2017;
14
67
5
8
0
9
11
6
9
5
12
8
3
8
6
3
16
4
0
0
2
4
6
8
10
12
14
16
18
Demographic characteristics of respondents
(n=20)
39
Irshad et al., 2017). The results of the antibacterial activity of the extracts and commercial
compounds are shown in Table 2.
Sarcina sp. was also tested against extracts of P. guineense for the first time in this study and the
results revealed that the extracts were active against the bacterium. Sarcina sp. was the most
sensitive bacterium to extracts of P. guineens. The hexane fruit and leaf extracts showed marked
inhibition zones against Sarcina sp. with an MIC as low as 19 µg/mL and was thus as effective as
piperine which gave an identical MIC value, this study therefore demonstrates that Piper guineense
could be explored as a lead to a new antibacterial drug for the treatment of human infections.
Sarcina sp. is an anaerobic Gram-positive bacterium belonging to the family Clostridiaceae.
Sarcina has mainly been characterized as the causative agent of abomasal bloat and death of
livestock (Lam-Himlin et al., 2011). There is still a debate whether Sarcina is pathogenic to
humans, although a number of cases of human disease, including gastric perforation,
emphysematous gastritis and gastric ulcer have been associated with Sarcina (Lam-Himlin et al.,
2011; De Meij et al., 2017). The hexane extracts of both P. guineense fruits and leaves contain
antibacterial piperamide alkaloids, which could be used as therapeutic agents in the treatment of
Sarcina infections and other stomach related problems, including gastric ulcers, diarrhoea and
foodborne diseases.
Table 2. Antibacterial effects of extracts of P. guineense, piperine and piperlongumine against
Gram-negative and Gram-positive bacteria. The results were obtained with the agar diffusion
method.
Extracts/
antibiotics
E.
aerogenes
Sarcina
sp.
S.
aureus
E.
coli
B.
cereus
P.
aeruginosa
S.
enterica
P.
mirabilis
Methanol fruit
extract
49.7
29.8
15.7
20.7
23.7
16.3
13.3
13.7
Chloroform
fruit extract
11.3
31.6
17.3
20.3
15.7
17.7
18.0
11.3
Hexane fruit
extract
34.7
37.6
17.3
NA
19.7
NA
12.7
11.7
Decoction fruit
NA
10.6
NA
NA
NA
NA
NA
NA
Maceration
fruit
NA
17.6
NA
NA
NA
NA
NA
NA
Ethanol fruit
extract
30.2
28.3
22.6
19.8
24.5
NT
21.7
16.3
Methanol leaf
extract
29.3
26.1
14.7
11.3
29.3
15.3
12.0
11.3
40
Chloroform
leaf extract
11.3
25.6
16.7
20.3
15.3
21.3
11.7
10.7
Hexane leaf
extract
17.8
33.6
15.0
NA
24.7
NA
NA
11.7
Decoction leaf
NA
10.6
NA
NA
NA
NA
NA
NA
Maceration leaf
NA
14.3
NA
NA
NA
NA
NA
NA
Ethanol leaf
extract
25.0
25.1
17.5
16.3
28.2
NT
16.2
13.8
Piperine
18.7
27.6
18.3
22.3
12.7
15.7
14.7
16.0
Piperlongumine
12.7
23.6
15.3
20.0
15.7
13.3
11.3
NA
Tetracycline
38.0
39.3
48.3
40.3
50.7
45.3
39.3
34.3
Rifampicin
45.3
41.6
49.3
49.7
54.3
55.7
55.7
40.7
Diameter of the zones of inhibition in mm. NA, not active; NT, not tested.
For Pseudomonas aeruginosa, the methanol and chloroform extracts of P guineense were the most
effective extracts against the bacterium with inhibition zones ranging from 15.3mm – 21.3mm
(publication II). Pseudomonas aeruginosa is a Gram-negative bacterium which most of the times
causes life-threatening nosocomial infections, and many combination therapy resistant strains have
been reported (Lister et al., 2009; Rasamiravaka et al., 2015). The chloroform extracts gave the
largest inhibition zone of 21.3 mm and the result shows that the chloroform extracts could contain
non-polar compounds that may be explored as antibacterial agents for multidrug-resistant
pathogens. The hexane, and water extracts were not active against Pseudomonas aeruginosa. The
present study shows that P. guineense could be a source of new antibacterial agents that will be
relevant for the treatment of nosocomial infections associated with Pseudomonas aeruginosa.
The cold water maceration and hot water decoctions of the extracts of P. guineense did not show
any inhibitory activity in most of the bacterial strains tested, this justifies the claim by traditional
healers that, the plant extracts are more active as a herbal remedy when prepared in mild alcohol
than in water decoctions and further correlates with the findings of Dada et al., 2013, which
reported that ethanol and hexane extracts of P. guineense are more effective than its water extracts.
The reason for water extracts lacking antibacterial activity could be attributed to the fact that the
bioactive compounds in publication (II) present in the other extracts, which are mostly piperamide
alkaloids, are not soluble in water.
Piperine and piperlongumine commercial compounds were active against most of the bacteria
investigated in our study, with piperlongumine showing the lowest MIC value of 9.7 µg/mL against
Sarcina sp. Piperine was found to be present in high concentrations in P. guineense leaves and
piperlongumine is known in many species of Piper, although we did not find it in the leaves of P.
41
guineense. Piperine and piperlongumine showed significant activity against E. coli with MIC values
of 19 and 39 µg/mL, respectively (II). From QTOF MS analysis conducted on the various extracts,
it could be seen that the extracts were rich in alkaloids, and thus, the inhibitory activities of these
extracts could be literally attributed to the presence of numerous piperamides present in P.
guineense in publication (II). Piperine showed significant activity against P. mirabilis with an
inhibition zone of 16.0 mm and a MIC value of 78 µg/mL and MBC value of 156 µg/mL
(publication (II). Thus, piperine must be responsible for a part of the good antibacterial activity of
the ethanol extracts of the seeds and leaves, since piperine was observed to be present in both
extracts according to our preliminary HPLC-DAD data. Thus, piperlongumine was not active
against P. mirabilis. Piperine and piperlongumine, were effective against E.coli with inhibition
zones of 22.3 mm and 20.0 mm respectively and MIC values of 19 and 39 µg/mL, respectively (II).
The extracts from P. guineense were susceptible to B. cereus with inhibition zones ranging from
15.3mm – 29.3mm (II). Based on the result obtained with this bacterium, the leaf extracts were
more active than the seed extracts. It could be possible that the leaf contains more antibacterial
compounds that could inhibit the growth of this particular bacterium. The hexane and chloroform
extracts were all active against the bacterium and the result demonstrates that the more polar
methanol and ethanol extracts as well as a hexane extract of the leaf contain some notable bioactive
compounds that may be responsible for the significant activity against B. cereus when compared
with the fruit extracts (II). This extract contains a multitude of piperamide alkaloids and their
isomers, of which many could be responsible for the good activity of the hexane leaf extract.
P. guineense extracts were active against S. aureus with inhibition zones ranging from 14.7 –
22.67mm and the largest inhibition zone was observed with an ethanol extract of the fruits
The results of this study on the antibacterial activity of the extracts of P. guineense against
Staphylococcus aureus justify the use of this plant in the treatment of common foodborne and other
infections caused by this bacterium. P. mirabilis was found to be resistant against most of the
extracts of P. guineense in the primary screening, using agar diffusion (II). From the result of the
study ethanol was found to be a good solvent for extracting compounds active against P. mirabilis.
For E. coli, Low MIC of 156 µg/mL was recorded in this study against E. coli for an ethanol
extract of the leaves of P. guineense. The methanol seed and leaf extracts of P. guineense gave large
inhibition zones of 20.7 mm and 20.3 mm respectively and these results suggest that standardized P.
guineense extracts could be used for possible alternative treatment against pathogenic multidrug-
resistant E. coli strains.
42
In this study, the extracts were active against Salmonella enterica. The ethanol fruit extract of P.
guineense showed the largest zone of inhibition of 21.7 mm for S. enterica and the ethanol leaf
extract showed a clear inhibition zone of 16.2 mm and a MIC of 78 µg/mL (II). The more polar
ethanol extracts contain antibacterial compounds that effectively inhibited the growth of this
bacterium. The chloroform, hexane and methanol extracts were moderately active also with
inhibition zones ranging from 18.0 – 11.7 mm.
5.3.2 Antifungal activity of Piper guineense (III)
The antifungal screening conducted in this study was done based on our ethnobotanical results on
the uses of P. guineense in West-African traditional medicine for the treatment of fungal diseases
(publication (III). In the investigation, pathogenic fungal strains which are known to be responsible
for part of the diseases treated by the traditional healers were chosen for the screening. Among all
the fungal strains selected, C. albicans is known to be the most significant human pathogenic
species of yeast that can cause serious fungal diseases in humans (Brown et al., 2014).
The results from the antifungal activity of the methanol, chloroform and n-hexane fractions of the
extracts of P. guineense on various candida strains (Candida albicans, Candida tropicalis, Candida
parapsilosis, and Candida glabrata) supports the use of the plant in the treatment of toilet
infections, sexually transmitted diseases and other microbial infections. This study recorded marked
MIC values, as low as 39 μg/mL were recorded against some of the fungal strains. The marked
antifungal activity recorded with the various extracts could be attributed to the presence of
piperamide alkaloids which were found to be present in the extracts in publication (II). The result
showed that the alkaloids present in the extracts of P. guineense is responsible for its antifungal
efficacy and these result correlated well with previous report that piperamides piperyline, 4,5-
dihydropiperylin and tetrahydropiperylin found in hexane fraction of P. arboreum showed
promising antifungal activities with tetrahydropiperyline having the lowest MIC of 15.6 μg/ml
against C. krusei, C. parapsilosis and C. neoformans (Regasini et al., 2009).The results of the
antifungal activity of the various extracts of P. guineense and the commercial compounds are
shown in Table 3. The result of the antifungal evaluation shows that the cold water maceration and
hot water decoctions of the fruit and leaf extracts of P. guineense does not have activity against the
fungal species tested in this study, thus P. guineense water extracts might be better to use in
combination with other medicinal plants as herbal remedy for treatment of fungal infections in
traditional medicine as seen in publication (III).
43
Table 3. Antifungal effects of extracts of P. guineense, piperine and piperlongumine against four
pathogenic strains of yeast and Cryptococcus neoformans. The results were obtained with the agar
diffusion method.
Plant extracts
and antibiotics
Candida
glabrata
Candida
albicans
Candida
parapsilosis
Candida
tropicalis
Cryptococcus
neoformans
Methanol fruit
extract
24.3
20.3
18.0
21.3
19.7
Chloroform
fruit extract
23.3
17.7
14.7
16.2
18.2
Hexane fruit
extract
203
14.3
15.3
11.0
14.3
Decoction fruit
NA
NA
NA
NA
NA
Maceration fruit
NA
NA
NA
NA
NA
Ethanol fruit
extract
NT
21.8
NT
19.8
22.2
Methanol leaf
extract
20.7
19.7
28.7
17.3
21.3
Chloroform leaf
extract
11.7
18.0
13.7
15.5
17.3
Hexane leaf
extract
13.7
21.7
17.8
12.0
11.7
Decoction leaf
NA
NA
NA
NA
NA
Maceration leaf
NA
NA
NA
NA
NA
Ethanol leaf
extract
NT
19.8
NT
18.2
23.8
Piperine
17.3
18.3
23.3
16.7
11.0
Piperlongumine
14.7
19.3
16.3
18.0
11.8
Itraconazole
16.3
25.3
29.7
26.2
29.7
Amphotericin B
34.3
35.7
43.7
29.3
38.0
The diameter of the zones of inhibition in mm. NA, not active; NT, not tested.
From the study, it was observed that the leaf extracts showed a different spectrum of activity
compared to the fruits. For example, the hexane leaf extract showed an inhibition zone of 21.7 mm
against C. albicans, whereas the hexane fruit extract only gave an IZ of 14.3 mm against this fungus
(III). The result could be supported by our ethnobotanical survey results, where the traditional
healers explained that the leaves of P. guineense are sometimes more active in the treatment of
some of the fungal infections and are therefore used more frequently than the fruit for some specific
symptoms of fungal infection and the results are in accordance with Rahmatullah et al., (2012), who
argued that the leaf extracts of medicinal plants are used more often in traditional medicine because
they are usually more effective than extracts made from other plant parts. The extracts were active
44
against C. albicans which is an opportunistic pathogenic fungus that is capable of causing serious
systemic infections in humans most especially individuals with compromised immune defences,
such as HIV/AIDS patients (Brown et al., 2014) (III). From the ethnobotanical survey, P.
guineense extracts when soaked in mild alcohol can be used for the treatment of vaginal candidiasis
which is often caused by C. albicans (III). Piperlongumine was found to be very active against C.
albicans with a MIC value of 39 µg/mL, while piperine gave a MIC of 78 µg/mL. The piperamide
alkaloids were also active against all the other tested fungal strains (III). The result obtained from
our study demonstrates that piperlongumine and piperine could be scaffolds for new natural plant
derived antifungal agents to combat multi-drug resistance in Candida strains. Marked growth
inhibitory activity was recorded against C. glabrata which is another significant human pathogenic
species of yeast. Previous research has shown that C. glabrata is a pathogenic fungal strain which is
capable of causing systemic infections which is often characterized with high mortality rate (Pfaller
et al., 2004; 2007). The largest inhibition zones were recorded with the methanol and chloroform
extracts against C. glabrata (IZ ranging from 24.3 mm to 20.7 mm). From the result, a low MIC
value of 0.48 µg/mL was recorded with amphotericin B against C. glabrata, and this result is in
agreement with the more frequent use of amphotericin B for the treatment of severe infections
caused by C. glabrata (Mario et al., 2012).
The efficacy of P. guineense extracts and its piperamide alkaloids on the yeast tested in this study
demonstrates that its extracts and alkaloids could be effectively utilized in combinations with
conventional antifungals for the treatment of fungal infections (III). The result further demonstrates
that the extracts and the piperamide compounds could be possible antifungal agents for the
discovery of a new antifungal drug for the treatment of systemic infections associated with the
tested fungal strains.
5.4 UHPLC/QTOF-MS results
From the present study, piperamides were found to be predominantly the major bioactive
compounds present in P. guineense extracts. The extracts contained piperamide compounds such as
piperine, dihydropiperine, piperylin, piperlonguminine, dihydropiperlonguminine, wisanine, and
dihydrowisanine. The results from the HPLC-DAD, UHPLC/Q-TOF MS and antimicrobial activity
indicates that these piperamides compounds identified in the extracts were responsible for the
antimicrobial efficacy of P. guineense and the result is in agreement with other previous authors
(Juliani et al., 2013; Besong et al., 2016). Altogether 18 compounds were identified in the extracts
(II). Among these compounds, the previously known piperamide alkaloids piperine,
45
dihydropiperine, piperylin, dihydropiperylin or piperlonguminine, dihydro-piperlonguminine,
wisanine, dihydrowisanine and various derivatives of piperine were identified and their mass
spectrometric data were compared to previous literature on piperamide alkaloids in Piper species
(Kotte et al., 2014; Rao et al., 2011; Adesina et al., 2003; Liu et al., 2015).
5.5 Synergistic effects of piperine/piperlongumine and antimicrobials
Piperine and piperlongumine were screened singly and in combination with conventional
antimicrobials to evaluate their synergetic, additive or antagonistic interactions against
Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans (IV). The aim was to
investigate the synergistic effects of piperine and piperlongumine when used singly and in
combination with conventional antimicrobials against Staphylococcus aureus, Pseudomonas
aeruginosa, and Candida albicans. Staphylococcus aureus, Pseudomonas aeruginosa, and Candida
albicans are human pathogens that are capable of causing systemic infection in humans. It has been
reported that 90 to 95% of Staphylococcus aureus strains are resistant to most conventional
antibiotics (Hemaiswarya et al., 2008; Kang et al., 2011). The Fractional inhibitory concentration
(FIC) index was used to determine the synergistic interaction between the commercial piperamides
and the antimicrobials. This method has been reported to be one of the most accurate methods to
determine synergistic interactions when the two inhibitors are studied in various combinations
(Tallarida, 2006; van Vuuren and Viljoen, 2011; van Vuuren et al., 2009). Synergistic effect is said
to be observed when the FIC index value of the compound of interest is less or equal to 0.5 (Kang et
al., 2011). For piperlongumine, there was synergistic effect at the ratio of 5:5 of rifampicin and
piperlongumine against Staphylococcus aureus (IV). This result demonstrates that the
pharmacological interaction between a bioactive compound and a conventional antimicrobial could
be affected by the ratio and concentration at which the two are combined. However antagonistic
interactions were observed between piperlongumine and tetracycline in all the ratio of combinations
tested against S. aureus. There is synergistic effect observed between piperine and rifampicin and
there is also synergistic effect between piperine and tetracycline against S. aureus. Rifampicin is an
antibiotic that is constantly used in the treatment of systemic bacterial infections in antimicrobial
therapy and this antibiotic is characterized with numerous adverse effects when used consecutively
for 10-14 days (Dhingra et al., 2004; Nageswari et al., 2018).
46
6. Conclusion
From this study, DryLab simulation program was successfully used to develop a rapid HPLC
method and an improved choice of mobile-phase for the analysis of P. guineense extracts.
Also from this study, it was observed that P. guineense extracts used for various medicinal purposes
and as herbal remedies, could be a potential lead to the discovery of a new antimicrobial drug. This
study demonstrated that extracts of P. guineense, enriched in alkaloids, have potent antibacterial
and antifungal activity against a panel of Gram-positive and Gram-negative bacteria, as well as
fungal strains, including significant human pathogens. Through the results obtained from this study
it has been established that alkaloids are the main bioactive constituents present in P. guineense
extracts. The water extracts were found to be devoid of these alkaloids, and thus inactive against
most of the studied bacterial and fungal strains tested in the study. The results revealed that P.
guineense alcohol, chloroform and hexane extracts are potent sources of piperamide compounds
which could help to combat bacterial and fungal infections, and might be used as an alternative to or
in combinations with synthetic antibiotics to address the problem of increasing microbial resistance
to synthetic antibiotics. Further research should be conducted to test the antibacterial and antifungal
mechanisms of action of piperamide compounds. It is also important to investigate the piperamide
compounds for their anti-biofilm activities, including quorum sensing. The inhibitory activity
observed with the various fractions and extracts against the tested bacterial and fungal strains
warrants further exploration of the bioactive compounds from P. guineense as molecular scaffolds
for new therapeutic agents in modern antimicrobial therapy. Further research could also be focused
on evaluating P. guineense extracts in combination with Xylopia aethiopica and other medicinal
plants for their antibacterial and antifungal effects. For these investigations, herbal formulations
identical to those used to treat infectious diseases by traditional healers in African traditional
medicine could be used.
Moreso, the antibacterial and antifungal efficacy of the methanol, ethanol, chloroform and n-hexane
extracts of the fruit and leaf of P. guineense, as well as piperine and piperlongumine on various
bacterial strains, C. albicans and non- albicans Candida strains supports the use of P. guineense in
the treatment of bacterial and fungal infections in traditional medicine and demonstrates that these
piperamide compounds can be utilized as therapeutic agents or scaffolds in the production of new
antibacterial and antifungal drugs to treat infectious diseases. From this study, it could be deduced
that the extracts and piperamide compounds exert promising antibacterial and antifungal properties
against pathogenic bacterial strains, Candida albicans and other non-albicans Candida strains.
Moreover, the extracts showed marked growth inhibitory profile against Cryptococcus neoformans
47
which is known to cause life-threatening meningitis in immunocompromised individuals. Based on
this current knowledge on alkaloids of P. guineense as antibacterial and antifungal agents, we
recommend that additional research should be done to evaluate the in vivo antibacterial and
antifungal properties of the extracts and piperamide compounds from P. guineense with some
animal models. Additional research is also needed to ascertain the antibacterial and antifungal
mechanism of action of the piperamide alkaloids.
The marked inhibitory activity recorded with piperine and piperlongumine against the bacterial
strains, C. albicans, and non-albicans Candida strains prompted us to study the piperamides in
combination with currently known conventional antibiotics, which the results of the study revealed
that piperine and piperlongumine could possibly enhance the effect of the currently available
antibacterial and antifungal drugs for the treatment of bacterial and fungal infections.
48
7. References
Abiala, M. A., Ayandeko, F. M., & Odebode, A. C. (2015). Antifungal effects of selected botanicals
on fungal pathogens of watermelon fruit. Archives of Phytopathology and Plant Protection, 48(7),
569-577.
Abila, B., Richens, A., & Davies, J. A. (1993). Anticonvulsant effects of extracts of the West
African black pepper, Piper guineense. Journal of Ethnopharmacology, 39(2), 113–117.
Abreu, A. C., Coqueiro, A., Sultan, A. R., Lemmens, N., Kim, H. K., Verpoorte, R., ... & Choi, Y.
H. (2017). Looking to nature for a new concept in antimicrobial treatments: Isoflavonoids from
Cytisus striatus as antibiotic adjuvants against MRSA. Scientific Reports, 7(1), 3777.
Adesina, S. K., Adebayo, A. S., & Gröning, R. (2003). New constituents of Piper guineense fruit
and leaf. Die Pharmazie, 58(6), 423-425.
Ajibesin, K. K., Bala, D. N., & Umoh, U. F. (2011). The use of medicinal plants to treat sexually
transmitted diseases in Nigeria: Ethnomedicinal survey of Niger Delta Region. International
Journal of Green Pharmacy (IJGP), 5(3).
Anyanwu, C. U., & Nwosu, G. C. (2014). Assessment of the antimicrobial activity of aqueous and
ethanolic extracts of Piper guineense leaves. Journal of Medicinal Plants Research, 8(10), 436–
440.
Asase, A., Hesse, D. N., & Simmonds, M. S. (2012). Uses of multiple plants prescriptions for
treatment of malaria by some communities in southern Ghana. Journal of Ethnopharmacology,
144(2), 448-452.
Asawalam, E. F. (2006). Insecticidal and repellent properties of Piper guineense seed oil extract for
the control of maize weevil, Sitophilus zeamais. Electronic Journal of Environmental, Agricultural
and Food Chemistry, 5(3), 1389-1394.
Besong, E. E., Balogun, M. E., Djobissie, S. F., Mbamalu, O. S., and Obimma, J. N. (2016). A
review of Piper guineense (African Black Pepper). International Journal of Pharmacy and
Pharmaceutical Research, 6 (1), 368-384.
49
Brown, A. J., Brown, G. D., Netea, M. G., & Gow, N. A. (2014). Metabolism impacts upon
Candida immunogenicity and pathogenicity at multiple levels. Trends in Microbiology, 22(11),
614-622.
Bezerra, D. P., Pessoa, C., de Moraes, M. O., Silveira, E. R., Lima, M. A. S., Elmiro, F. J. M., and
Costa-Lotufo, L. V. (2005). Antiproliferative effects of two amides, piperine and piplartine, from
Piper species. Zeitschrift für Naturforschung C, 60(7-8), 539-543.
Bezerra, D. P., Castro, F. O., Alves, A. P. N. N., Pessoa, C., Moraes, M. O., Silveira, E. R., ...and
Costa-Lotufo, L. V. (2006). In vivo growth-inhibition of Sarcoma 180 by piplartine and piperine,
two alkaloid amides from Piper. Brazilian Journal of Medical and Biological Research, 39(6), 801–
807.
Bezerra, D. P., Castro, F. O. D., Alves, A. P. N., Pessoa, C., Moraes, M. O. D., Silveira, E. R., and
Lima, M. W. (2008). In vitro and in vivo antitumor effect of 5‐FU combined with piplartine and
piperine. Journal of Applied Toxicology, 28(2), 156–163.
Chonpathompikunlert, P., Wattanathorn, J., & Muchimapura, S. (2010). Piperine, the main alkaloid
of Thai black pepper, protects against neurodegeneration and cognitive impairment in animal model
of cognitive deficit like condition of Alzheimer’s disease. Food and Chemical Toxicology, 48(3),
798-802.
Cheikhyoussef, A., Shapi, M., Matengu, K., & Ashekele, H. M. (2011). Ethnobotanical study of
indigenous knowledge on medicinal plant use by traditional healers in Oshikoto region, Namibia.
Journal of Ethnobiology and Ethnomedicine, 7(1), 10.
Cockerill, F.R., Wikler, M., Bush, K., Dudley, M., Eliopoulos, G., Hardy, D., (2012). Clinical and
Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing:
twenty-second informational supplement. Approved Standard—Ninth Edition. 950 West Valley
Road, Suite 2500, Wayne, Pennsylvania 19087, USA.
50
Dada, A. A., Ifesan, B. O. T., & Fashakin, J. F. (2013). Antimicrobial and antioxidant properties of
selected local spices used in “Kunun” beverage in Nigeria. Acta Scientiarum Polonorum
Technologia Alimentaria, 12(4), 373-378.
De Meij, T. G., van Wijk, M. P., Mookhoek, A., & Budding, A. E. (2017). Ulcerative Gastritis and
esophagitis in two Children with Sarcina ventriculi Infection. Frontiers in Medicine, 4, 145.
Dhingra, V. K., Rajpal, S., Aggarwal, N., Aggarwaln, J. K., Shadab, K., & Jain, S. K. (2004).
Adverse drug reactions observed during DOTS. The Journal of Communicable Diseases, 36(4),
251-259.
Dzoyem, J. P., Tchuenguem, R. T., Kuiate, J. R., Teke, G. N., Kechia, F. A., & Kuete, V. (2014). In
vitro and in vivo antifungal activities of selected Cameroonian dietary spices. BMC Complementary
and Alternative Medicine, 14(1), 58.
Ekundayo, O., Laakso, I., Adegbola, R. M., Oguntimein, B., Sofowora, A., & Hiltunen, R. (1988).
Essential oil constituents of Ashanti pepper (Piper guineense) fruits (Berries). Journal of
Agricultural and Food Chemistry, 36(5), 880-882.
Ene-Obong, H., Onuoha, N., Aburime, L., & Mbah, O. (2018). Chemical composition and
antioxidant activities of some indigenous spices consumed in Nigeria. Food Chemistry, 238, 58–64.
Fyhrquist, P., Laakso, I., Marco, S. G., Julkunen-Tiitto, R., & Hiltunen, R. (2014).
Antimycobacterial activity of ellagitannin and ellagic acid derivate rich crude extracts and fractions
of five selected species of Terminalia used for treatment of infectious diseases in African traditional
medicine. South African Journal of Botany, 90, 1-16.
Freiesleben, S. H., Soelberg, J., and Jäger, A. K. (2015). Medicinal plants used as excipients in the
history in Ghanaian herbal medicine. Journal of Ethnopharmacology, 174, 561-568.
Gbekley, H. E., Katawa, G., Karou, S. D., Anani, S., Tchadjobo, T., Ameyapoh, Y., and Simpore, J.
(2017). Ethnobotanical study of plants used to treat asthma in the maritime region in Togo. African
Journal of Traditional, Complementary, and Alternative Medicines, 14(1), 196.
51
Hemaiswarya, S., Kruthiventi, A. K., & Doble, M. (2008). Synergism between natural products and
antibiotics against infectious diseases. Phytomedicine, 15(8), 639-652.
Ibrahim, J. A., Muazzam, I., Jegede, I. A., & Kunle, O. F. (2010). Medicinal plants and animals sold
by the Yan-Shimfidas of Sabo Wuse in Niger State, Nigeria. African Journal of Pharmacy and
Pharmacology, 4(6), 386-394.
Irshad, S., Ashfaq, A., Muazzam, A., & Yasmeen, A. (2017). Antimicrobial and Anti-Prostate
Cancer Activity of Turmeric (Curcuma longa L.) and Black Pepper (Piper nigrum L.) used in
Typical Pakistani Cuisine. Pakistan Journal of Zoology, 49(5).
Iweala, E. E. J. (2015). Anti-Cancer and Free Radical Scavenging Activity of Some Nigerian Food
Plants In vitro. International Journal of Cancer Research, 11(1), 41-51.
Jha, P., Kim, C. M., Kim, D. M., Chung, J. H., Yoon, N. R., Jha, B., & Jeon, D. Y. (2016).
Transmission of Enterobacter aerogenes septicemia in healthcare workers. SpringerPlus, 5(1),
1397.
Juliani Rodolfo, H., Koroch, A. R., Giordano, L., Amekuse, L., Koffa, S. J., Asante-Dartey, S. E., &
Simon, J. E. (2013). Piper guineense (Piperaceae): Chemistry, Traditional Uses and Functional
Properties of West African Black Pepper. African Natural Plant Products. Discoveries and
Challenges in Chemistry. In Health and Nutrition. ACS Symposium Series (Vol. 2, p. 1127).
Kamtchouing, P., Mbongue, G. Y. F., Dimo, T., Watcho, P., Jatsa, H. B., and Sokeng, S. D. (2002).
Effects of Aframomum melegueta and Piper guineense on sexual behavior of male rats. Behavioural
Pharmacology, 13(3), 243–247.
Kang, H. K., Kim, H. Y., & Cha, J. D. (2011). Synergistic effects between silibinin and antibiotics
on methicillin‐resistant Staphylococcus aureus isolated from clinical specimens. Biotechnology
Journal, 6(11), 1397-1408.
Konning, G. H., Agyare, C., and Ennison, B. (2004). Antimicrobial activity of some medicinal
plants from Ghana. Fitoterapia, 75(1), 65–67.
52
Kotte, S. C. B., Dubey, P. K., & Murali, P. M. (2014). Identification and characterization of stress
degradation products of piperine and profiling of a black pepper (Piper nigrum L.) extract using
LC/Q-TOF-dual ESI-MS. Analytical Methods, 6(19), 8022-8029.
Lam-Himlin, D., Tsiatis, A. C., Montgomery, E., Pai, R. K., Brown, J. A., Razavi, M., ... & Anders,
R. A. (2011). Sarcina organisms in the gastrointestinal tract: a clinicopathologic and molecular
study. The American Journal of Surgical Pathology, 35(11), 1700.
Lister, P. D., Wolter, D. J., & Hanson, N. D. (2009). Antibacterial-resistant Pseudomonas
aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance
mechanisms. Clinical Microbiology Reviews, 22(4), 582-610.
Liu, H. L., Luo, R., Chen, X. Q., Ba, Y. Y., Zheng, L., Guo, W. W., & Wu, X. (2015). Identification
and simultaneous quantification of five alkaloids in Piper longum L. by HPLC–ESI-MS and
UFLC–ESI-MS/MS and their application to Piper nigrum L. Food Chemistry, 177, 191-196.
Mario, D. A. N., Denardi, L. B., Bandeira, L. A., Antunes, M. S., Santurio, J. M., Severo, L. C., &
Alves, S. H. (2012). The activity of echinocandins, amphotericin B and voriconazole against
fluconazole-susceptible and fluconazole-resistant Brazilian Candida glabrata isolates. Memórias do
Instituto Oswaldo Cruz, 107(3), 433-436.
Mbongue, F. G., Kamtchouing, P., Essame, O. J., Yewah, P. M., Dimo, T., and Lontsi, D. (2005).
Effect of the aqueous extract of dry fruits of Piper guineense on the reproductive function of adult
male rats. Indian Journal of Pharmacology, 37(1), 30.
Mgbeahuruike E.E, Yrjönen T, Vuorela H, and Holm Y. (2017). Bioactive compounds from
medicinal plants: focus on Piper species. South African Journal of Botany, 112, 54-69.
Maroyi, A. (2013). Traditional use of medicinal plants in south-central Zimbabwe: review and
perspectives. Journal of Ethnobiology and Ethnomedicine, 9(1), 31.
Nageswari, A. D., Rajanandh, M. G., Uday, M. K. R. A., Nasreen, R. J., Pujitha, R. R., &
Prathiksha, G. (2018). Effect of rifampin with bio-enhancer in the treatment of newly diagnosed
sputum positive pulmonary tuberculosis patients: A double-center study. Journal of Clinical
Tuberculosis and Other Mycobacterial Diseases, 12, 73-77.
53
Ngane, A. N., Biyiti, L., Bouchet, P. H., Nkengfack, A., and Zollo, P. A. (2003). Antifungal activity
of Piper guineense of Cameroon. Fitoterapia, 74(5), 464-468.
Ngarivhume, T., van’t Klooster, C. I., de Jong, J. T., & Van der Westhuizen, J. H. (2015).
Medicinal plants used by traditional healers for the treatment of malaria in the Chipinge district in
Zimbabwe. Journal of Ethnopharmacology, 159, 224-237.
Nwozo, S. O., Lewis, Y. T., and Oyinloye, B. E. (2017). The effects of Piper guineense versus
Sesamum indicum aqueous extracts on lipid metabolism and antioxidants in hypercholesterolemic
rats. Iranian Journal of Medical Sciences, 42(5), 449.
Nyiredy S. (2002) Planar chromatographic method development using the PRISMA optimization
system and flow charts. Journal of Chromatographic Science, 40, 553-563.
Obodozie, O. O., Ameh, S. J., Afolabi, E. K., Oyedele, E. O., Ache, T. A., Onanuga, C. E., ... &
Inyang, U. S. (2010). A normative study of the components of niprisan—an herbal medicine for
sickle cell anemia. Journal of Dietary Supplements, 7(1), 21–30.
Oboh, G., Ademosun, A. O., Odubanjo, O. V., and Akinbola, I. A. (2013). Antioxidative properties
and inhibition of key enzymes relevant to type-2 diabetes and hypertension by essential oils from
black pepper. Advances in Pharmacological Sciences, 2013, 926047.
Ode, O. J., Saka, S., & Oladele, G. M. (2011). The global relevance of traditional medicine and
herbal plants, the nigerian perspective. International Journal of Applied Biology and
Pharmaceutical Technology, 2(4), 280-289.
Oguntibeju, O. O. (2018). Medicinal plants with anti-inflammatory activities from selected
countries and regions of Africa. Journal of inflammation research, 11, 307.
Ogunniran, K. O. (2009). Antibacterial effects of extracts of Ocimum gratissimum and piper
guineense on Escherichia coli and Staphylococcus aureus. African Journal of Food Science, 3(3),
77–81.
54
Olorunnisola, O. S., Adetutu, A., Balogun, E. A., & Afolayan, A. J. (2013). Ethnobotanical survey
of medicinal plants used in the treatment of malarial in Ogbomoso, Southwest Nigeria. Journal of
Ethnopharmacology, 150(1), 71-78.
Okeke, M. I., Iroegbu, C. U., Jideofor, C. O., Okoli, A. S., & Esimone, C. O. (2001). Anti-microbial
activity of ethanol extracts of two indigenous Nigerian spices. Journal of Herbs, Spices &
Medicinal Plants, 8(4), 39-46.
Okigbo, R., & Igwe, D. (2007). Antimicrobial effects of Piper guineense ‘Uziza’and Phyllantus
amarus ‘Ebe-benizo’on Candida albicans and Streptococcus faecalis. Acta Microbiologica et
Immunologica Hungarica, 54(4), 353–366.
Okon, E. E., Chibuzor, E. F., Christian, O. E., Nsikan, U. M., and Francis, A. M. (2013). In vitro
antioxidant and nitric oxide scavenging activities of Piper guineense seeds. Global Journal of
Research on Medicinal Plants & Indigenous Medicine, 2(7), 475.
Olonisakin, A., Oladimeji, M. O., & Lajide, L. (2006). Chemical composition and antibacterial
activity of steam distilled oil of Ashanti pepper (Piper guineense) fruits (berries). Electronic
Journal of Environmental, Agricultural and Food Chemistry, 5, 1531-1535.
Oyemitan, I. A., Olayera, O. A., Alabi, A., Abass, L. A., Elusiyan, C. A., Oyedeji, A. O., and
Akanmu, M. A. (2015). Psychoneuropharmacological activities and chemical composition of
essential oil of fresh fruits of Piper guineense (Piperaceae) in mice. Journal of Ethnopharmacology,
166, 240–249.
Oyinloye, B. E., Osunsanmi, F. O., Ajiboye, B. O., Ojo, O. A., & Kappo, A. P. (2017). Modulatory
Effect of Methanol Extract of Piper guineense in CCl4-Induced Hepatotoxicity in Male Rats.
International Journal of Environmental Research and Public Health, 14(9), 955.
Parmar, V. S., Jain, S. C., Bisht, K. S., Jain, R., Taneja, P., Jha, A., and Boll, P. M. (1997).
Phytochemistry of the genus Piper. Phytochemistry, 46(4), 597-673.
Pfaller, M. A., & Diekema, D. J. (2007). Epidemiology of invasive candidiasis: a persistent public
health problem. Clinical Microbiology Reviews, 20(1), 133-163.
55
Pfaller, M. A., & Diekema, D. J. (2004). Rare and emerging opportunistic fungal pathogens:
concern for resistance beyond Candida albicans and Aspergillus fumigatus. Journal of Clinical
Microbiology, 42(10), 4419–4431.
Regasini, L. O., Cotinguiba, F., Morandim, A. D. A., Kato, M. J., Scorzoni, L., Mendes-Giannini,
M. J., & Furlan, M. (2009). Antimicrobial activity of Piper arboreum and Piper tuberculatum
(Piperaceae) against opportunistic yeasts. African Journal of Biotechnology, 8(12).
Rao, V. R. S., Raju, S. S., Sarma, V. U., Sabine, F., Babu, K. H., Babu, K. S., & Rao, J. M. (2011).
Simultaneous determination of bioactive compounds in Piper nigrum L. and a species comparison
study using HPLC-PDA. Natural Product Research, 25(13), 1288-1294.
Rahmatullah, M., Hossan, S., Khatun, A., Seraj, S., & Jahan, R. (2012). Medicinal plants used by
various tribes of Bangladesh for treatment of malaria. Malaria Research and Treatment, 2012,
371798.
Rasamiravaka, T., Labtani, Q., Duez, P., & El Jaziri, M. (2015). The formation of biofilms by
Pseudomonas aeruginosa: a review of the natural and synthetic compounds interfering with control
mechanisms. BioMed Research International, 759348, 1–17.
Salih, E. Y. A., Kanninen, M., Sipi, M., Luukkanen, O., Hiltunen, R., Vuorela, H., & Fyhrquist, P.
(2017). Tannins, flavonoids and stilbenes in extracts of African savanna woodland trees Terminalia
brownii, Terminalia laxiflora and Anogeissus leiocarpus showing promising antibacterial potential.
South African Journal of Botany, 108, 370-386.
Scott, I. M., Puniani, E., Jensen, H., Livesey, J. F., Poveda, L., Sánchez-Vindas, P., and Arnason, J.
T. (2005). Analysis of Piperaceae germplasm by HPLC and LCMS: a method for isolating and
identifying unsaturated amides from Piper spp extracts. Journal of Agricultural and Food
Chemistry, 53(6), 1907–1913.
Subramani, R., Narayanasamy, M., & Feussner, K. D. (2017). Plant-derived antimicrobials to fight
against multi-drug-resistant human pathogens. 3 Biotech, 7(3), 172.
56
Tallarida, R. J. (2006). An overview of drug combination analysis with isobolograms. Journal of
Pharmacology and Experimental Therapeutics, 319(1), 1-7.
Taulavuori, K., Julkunen-Tiitto, R., Hyöky, V., & Taulavuori, E. (2013). Blue mood for superfood.
Natural Product Communication, 8(6), 791-4.
Tekwu, E. M., Askun, T., Kuete, V., Nkengfack, A. E., Nyasse, B., Etoa, F. X., and Beng, V. P.
(2012). Antibacterial activity of selected Cameroonian dietary spices ethno-medically used against
strains of Mycobacterium tuberculosis. Journal of Ethnopharmacology, 142(2), 374–382.
Uhegbu, F. O., Imo, C., & Ugbogu, A. E. (2015). Effect of aqueous extract of Piper guineense seeds
on some liver enzymes, antioxidant enzymes and some hematological parameters in Albino rats.
International Journal of Plant Science and Ecology, 1, 167–171.
Udoh, F. V. (1999). Uterine muscle reactivity to repeated administration and phytochemistry of the
leaf and seed extracts of Piper guineense. Phytotherapy Research, 13(1), 55–58.
Umoh, I., Oyebadejo, S., Bassey, E. O., & Udoh, N. (2013). Histomorphological study of the effect
of chronic consumption of Abelmoschus esculentus and Piper guineense on the gastric mucosa of
albino wistar rats. International Journal of Pharmaceutical Research and Allied Sciences, 2, 31–37.
Van Vuuren, S. F., Suliman, S., & Viljoen, A. M. (2009). The antimicrobial activity of four
commercial essential oils in combination with conventional antimicrobials. Letters in Applied
Microbiology, 48(4), 440-446.
Van Vuuren, S., & Viljoen, A. (2011). Plant-based antimicrobial studies–methods and approaches
to study the interaction between natural products. Planta Medica, 77(11), 1168-1182.
World Health Organization. (2001). Legal status of traditional medicine and complementary (No.
WHO/EDM/TRM/2001.2). Geneva: World Health Organization.
