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Beschreibung

Both Asia and Africa are home to many plants that can be used for the treatment of many diseases and their medicinal properties are gaining interest in western societies. Medicinal plants from Asia and Africa are used for their healing abilities and also have a symbolic meaning in communities.The importance of traditional autochthonous plant remedies plays a crucial role in the health of millions of people of these two continents. Even today, traditional medicine represents the dominant medical system for millions of people showing a significant impact on health care practices. Therefore, traditional operators still represent a vital part of regional healthcare systems. For this reason, pharmaceutical industries consider traditional medicine as a source for the identification of bioactive compounds that can be used in the preparation of synthetic drugs.Biologically Active Natural Products from Asia and Africa: A Selection of Topics guides the reader toinformation about new natural products from these regions and the different ways to use them to treat or alleviate many of the most common diseases. The volume presents nine topics covering a number of facets of natural product medicine including: - pharmaceutical analysis of anti-diabetic herbal medicines from Bangladesh and local retailers- caffeine intake and the risk of female infertility- pharmaceutical analysis of Urena sinuata (bur mallow)- anti-CHIKV activities of diterpenes and their derivatives- anti-inflammatory nanogel for the treatment of psoriasis- antlithiatic properties of Moroccan medicinal plants- ethnobotanic, phytochemical and biological activities of Aristolochia longa L. (pipevine)- wound healing potential of combined extracts of stem bark and leaves of sphenocentrum jollyanum (an African shrub) This is a handy reference for specialists and R&D experts in pharmaceutical chemistry who wish to be informed about current knowledge on developing natural remedies in Asia and Africa.

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Table of Contents
BENTHAM SCIENCE PUBLISHERS LTD.
End User License Agreement (for non-institutional, personal use)
Usage Rules:
Disclaimer:
Limitation of Liability:
General:
PREFACE
List of Contributors
Biological and Physical Contaminants in Anti-Diabetic Herbal Medicines of Bangladesh
Abstract
INTRODUCTION
Methods
Study Area and Sample Collection
Determination of pH
Microbiological Analysis
Preparation of Culture Media
Isolation and Identification of Microorganisms
Total Aerobic Bacterial Count and Total Coliform Count
Escherichia coli 0157
Escherichia coli
Salmonella spp.
Listeria spp.
Yeasts and Molds Count (YMC)
Toxic Metal Analysis
Results
Microbial Contamination
Metal Toxicity
Discussion
Biological Contamination (Microbes)
Physical Contamination (Metal Toxicity)
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Quantification and Health Safety Assessment of Some Toxic Metals in Anti-Diabetic Herbal Preparations Collected from Local Retailers Using the XRF Analytical Tool
Abstract
INTRODUCTION
METHODS
Study Area and Sample Collection
Sample Preparation and Analysis
RESULTS AND DISCUSSION
CONCLUSION
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Caffeine Intake and the Risk of Female Primary Infertility: An Evidence-Based Case Report
Abstract
CASE
INTRODUCTION
Methods
Results
Discussion
CONCLUSION
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
An Overview of Urena sinuata: Phytochemistry and Pharmacological Activities
Abstract
INTRODUCTION
RESEARCH METHODOLOGY
FINDINGS
Phytochemicals of U. Sinuata
Pharmacological Activities of U. Sinuata
Antioxidant Activity
Anti-Diarrhoeal Activity
Anti-Nociceptive Activity
Anti-Inflammatory Activity
Anti-Pyretic Activity
Insecticidal and Insect-Repellent Activities
Thrombolytic Activity
Sedative, Anxiolytic, and Analgesic Activities
TOXICOLOGICAL PROFILE
DISCUSSION
SUMMARY AND PERSPECTIVES
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Anti-CHIKV Activities of Diterpenes and Their Derivatives
Abstract
INTRODUCTION
SEARCH STRATEGY
FINDINGS
CONCLUSION AND FUTURE PERSPECTIVES
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Design Development and Evaluation of Anti-Inflammatory Nanogel For The Treatment of Psoriasis
Abstract
INTRODUCTION
MATERIALS AND METHODS
Collection of Plant Material
Extraction of Plant Material
FORMULATIONS OF NANOGEL
Preparation of Drug-Loaded Nanodispersions
Preparation of Nanogel
EVALUATION OF DEVELOPED FORMULATION
Appearance
Determination of pH
Determination of Homogeneity
Viscosity
Particle Size
Spreadability
Skin Irritancy Test
RESULTS AND DISCUSSION
Particle Size Determination
Zeta Potential Measurements
Stability Studies
IN-VIVO ANTI-INFLAMMATORY ACTIVITY
Animals
Carrageenan-Induced Mice Paw Oedema Method
Statistical Analysis
RESULTS AND DISCUSSION
DISCUSSION AND CONCLUSION
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Antilithiatic Properties of Moroccan Medicinal Plants and Mechanism Insights of their Phytochemicals
Abstract
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
Ethnobotany of Antilithiasic Medicinal Plants Used in Morocco
Experimental Methods to Evaluate The Antilithiatic Activity
In Vitro Methods
In Vivo Methods
ANTILITHIATIC MECHANISM OF PHYTOMOLECULES
Changing Urine Parameters
Diuretic
Influence on The Crystallization Process
Cell Surface Adhesion
Antioxidant Effect
CONCLUSION
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Ethnobotanic, Phytochemical, and Biological Activities of Aristolochia longa L.: A Review
Abstract
INTRODUCTION
CLASSIFICATION AND BOTANICAL DESCRIPTION
Ethnomedicobothany
Phytochemistry of A. longa
Major Compounds, Including Phenols, Flavonoids, Alkaloids, Saponins
Fatty Acid Composition
PHARMACOLOGICAL AND BIOLOGICAL ACTIVITIES OF A. LONGA COMPOUNDS
Anti-tumor Activities
Antibacterial and Antifungal Activities
Antidiabetic Activities
Antioxidant Activities
Anti-inflammatory Activities
Immunostimulatory Activities
Toxicity Effect
CONCLUSION
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Wound Healing Potential of Combined Extracts of Stem Bark and Leaves of Sphenocentrum Jollyanum: A Classical Factorial Design Model Approach
Abstract
INTRODUCTION
MATERIALS AND METHODS
Collection and Processing of Plant Materials
Preparation of The Plant Extracts
Phytochemical Analysis of Plant Extracts
Experimental Animals and Test Groups
Surgical Infliction of Excision Wound
Wound Treatment
Data Collection and Analysis
Statistical Analysis
RESULTS
Phytochemical Constituents of Plant Extracts
Wound Closure Effects of Plant Extracts
Effects of Combined Extracts on Wound Healing Activities
Wound Surface pH Profile
DISCUSSION
CONCLUSION
CONSENT FOR PUBLICATION
CONFLICT OF INTEREST
ACKNOWLEDGEMENTS
REFERENCES
Biologically Active Natural Products from Asia and Africa: A Selection of Topics
Edited by
Anna Capasso
Department of Pharmaceutical Sciences
University of Salerno, 84084 Fisciano SA
Italy

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PREFACE

Both Asia and Africa have many plants that can be used for medicinal purposes: these medicinal plants are used in the treatment of many diseases and their uses and effects are of growing interest in western societies. Medicinal plants from Asia and Africa are not only used and chosen for their healing abilities, but also for a symbolic and spiritual meaning.

The importance of traditional autochthonous plant remedies plays a crucial role in the health of millions of people both in Asia and in Africa. Even today, traditional medicine represents the dominant medical system for millions of people showing a significant impact on health care practices. Therefore, traditional operators still represent a vital part of their health system. For this reason, even the pharmaceutical industries consider traditional medicine as a source for the identification of bioactive agents that can be used in the preparation of synthetic drugs.

This book will guide you to discover new natural products from Asia and Africa and the different ways to use them to treat or alleviate many of the most common diseases.

Anna Capasso Department of Pharmaceutical Sciences University of Salerno, 84084 Fisciano SA Italy

List of Contributors

Zaifar A., Faculty of MedicineUniversitas IndonesiaJakartaIndonesiaYelmate A.A., School of PharmacyS.R.T.M. UniversityNanded, MaharashtraIndiaBouyahya Abdelhakim, Department of Biology, Faculty of ScienceGenomic Center of Human Pathologies, Faculty of Medicine, Laboratory of Human Pathologies Biology, Mohammed V UniversityRabatMoroccoFarooque Akhter, Department of ChemistryUniversity of RajshahiRajshahiBangladeshYahyaoui El Idrissi Amina El, Department of Biology, Faculty of ScienceGenomic Center of Human Pathologies, Faculty of Medicine, Laboratory of Human Pathologies Biology, Mohammed V UniversityRabatMoroccoKhouchlaa Aya, Department of Biology, Faculty of ScienceGenomic Center of Human Pathologies, Faculty of Medicine, Laboratory of Human Pathologies Biology, Mohammed V UniversityRabatMoroccoNnadi Charles O., Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical SciencesUniversity of Nigeria Nsukka410001 NsukkaNigeriaUgwu Chigozie L., Department of Statistics, Faculty of Physical SciencesUniversity of Nigeria Nsukka410001 NsukkaNigeriaOnah Chinwe M., Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical SciencesUniversity of Nigeria Nsukka410001 NsukkaNigeriaNabila E.N., Faculty of MedicineUniversitas IndonesiaJakartaIndonesiaVincent L.D., Faculty of MedicineUniversitas IndonesiaJakartaIndonesiaLouisa M., Department of Pharmacology and Therapeutics, Faculty of MedicineUniversitas IndonesiaJakartaIndonesiaRahman M. Safiur, Atmospheric and Environmental Chemistry LaboratoryChemistry Division, Atomic Energy Center4-Kazi Nazrul Islam Avenue, Dhaka-1000BangladeshTijane M'hamed, Department of Biology, Faculty of ScienceGenomic Center of Human Pathologies, Faculty of Medicine, Laboratory of Human Pathologies Biology, Mohammed V UniversityRabatMoroccoParveen Mahfuza, Department of Environmental Science and Disaster ManagementDaffodil International UniversityDhaka-1207BangladeshAsraf Md. Ali, Department of ChemistryUniversity of RajshahiRajshahiBangladeshRahman Md. Matiur, Department of PharmacyRanada Prasad Shaha UniversityNarayanganj-1400BangladeshFessikh Mereym El, Department of Biology, Faculty of Science, Genomic Center of Human PathologiesFaculty of Medicine, Laboratory of Human Pathologies Biology, Mohammed V UniversityRabatMoroccoIslam Muhammad Torequl, Department of Pharmacy, Life Science FacultyBangabandhu Sheikh Mujibur Rahman Science and Technology UniversityGopalganj (Dhaka)-8100BangladeshKusuma N.M.P, Faculty of MedicineUniversitas IndonesiaJakartaIndonesiaIslam Nazmul, Department of General Educational DevelopmentDaffodil International UniversityDhakaBangladeshDepartment of Nutrition and Food TechnologyJashore University of Science and TechnologyJashoreBangladeshBhoumik Nikhil C., Wazed Miah Science Research Center, Jahangirnagar University, SavarDhakaBangladeshFaruque Omar, Department of Nutrition and Food TechnologyJashore University of Science and TechnologyJashoreBangladeshGundewar P., School of PharmacyS.R.T.M. UniversityNanded, MaharashtraIndiaRay Pranta, Department of Pharmacy, Life Science FacultyBangabandhu Sheikh Mujibur Rahman Science and Technology UniversityGopalganj (Dhaka)-8100BangladeshMoon R.S., School of PharmacyS.R.T.M. UniversityNanded, MaharashtraIndiaHossain Rajib, Department of Pharmacy, Life Science FacultyBangabandhu Sheikh Mujibur Rahman Science and Technology UniversityGopalganj (Dhaka)-8100BangladeshKanti Rajib, Department of Nutrition and Food TechnologyJashore University of Science and TechnologyJashoreBangladeshZamir Rausan, Department of ChemistryUniversity of RajshahiRajshahiBangladeshNareswari S.S.K., Department of Pharmacology and Therapeutics, Faculty of MedicineUniversitas IndonesiaJakartaIndonesiaSarker Shipra, Department of Nutrition and Food TechnologyJashore University of Science and TechnologyJashoreBangladeshAhmed Sunzid, Centre for Advanced Research in ScienceUniversity of DhakaDhakaBangladeshObonga Wilfred O., Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical SciencesUniversity of Nigeria Nsukka410001 NsukkaNigeriaBakri Youssef, Department of Biology, Faculty of ScienceGenomic Center of Human Pathologies, Faculty of Medicine, Laboratory of Human Pathologies Biology, Mohammed V UniversityRabatMorocco

Biological and Physical Contaminants in Anti-Diabetic Herbal Medicines of Bangladesh

Rausan Zamir1,*,Nazmul Islam2,5,Sunzid Ahmed3,Md. Ali Asraf1,Nikhil C. Bhoumik4,Omar Faruque5,Akhter Farooque1
1 Department of Chemistry, University of Rajshahi, Rajshahi, Bangladesh
2 Department of General Educational Development, Daffodil International University, Dhaka, Bangladesh
3 Centre for Advanced Research in Science, University of Dhaka, Dhaka, Bangladesh
4 Wazed Miah Science Research Center, Jahangirnagar University, Savar, Dhaka, Bangladesh
5 Department of Nutrition and Food Technology, Jashore University of Science and Technology, Jashore, Bangladesh

Abstract

The present study evaluated biological and physical contamination in terms of microbes and toxic metal, respectively, in eight antidiabetic herbal medicines (ADHMs) from different markets in Dhaka City, Bangladesh. Coliform, E. coli, Salmonella spp. and Listeria spp. were absent in all ADHMs. However, aerobic bacterial count of all the samples of yeasts and molds in some samples fails to satisfy safe limits set by different regulatory standards. Among the nine metals [Copper (Cu), Zinc (Zn), Lead (Pb), Manganese (Mn), Chromium (Cr), Iron (Fe), Cadmium (Cd), Nickel (Ni), and Arsenic (As)] investigated, Cu, Zn, Pb, Cr, Ni content was in safe limit according to different pharmacopoeia and WHO guidelines. Among all the regulatory authorities, only the Health Sciences Authority (HSA), Singapore claims the Cd content is above the permissible limit in all the samples except ADHM-4. Chinese pharmacopoeia restricts the use of ADHM-1, ADHM-2 and ADHM-8 because of unacceptable arsenic (As) contamination. All the targeted antidiabetic herbal medicines (ADHMs) were found to retain an unacceptable level of Mn, ranging from 0.44±0.01 to 4.17±0.03 ppm. Metals contamination poses potential risks to human health and regulatory authorities not only should impose a restriction on the use of the medicines but also direct guidelines to keep the drugs safe.

Keywords: ADHMs, Human health and drug safety, Metal toxicity, Microbes.
*Corresponding author Rausan Zamir: Department of Chemistry, University of Rajshahi, Rajshahi, Bangladesh; Tel: +88-01711-705705; Fax: +88-0721-750064; E-mail: [email protected]

INTRODUCTION

Sometimes the conventional medicinal system fails to successfully treat some illnesses [1-3]. This inability results in a negative medical encounter, which badly affects the doctor-patient relationship [4-6]. As a result, being repulsive with conventional medicine and being attracted to some sort of values and beliefs, also referred to as postmodern philosophy, a fraction of people turns to the alternate medicinal system [4, 7, 8]. In Bangladesh, people are not required to pay extra money to the physician for a prescription and for traditional treatment, no practitioner suggests diagnosis for the investigation disorder related to physic. Therefore, people pay only for traditional drugs. Moreover, the high cost and side effects of most modern drugs shifted consumer’s attention from conventional to herbal medicines [9]. Therefore, the uses of herbal medicines are increasing day by day throughout the world [10]. Consumer awareness has increased with the work of ad agencies who are airing undue respectability and credibility of herbal products on television and radio programs [11, 12]. These advertisements aim to attract the different age groups of people with their selective presentation. A child requires healthy growth and development. In his youth, the man requires to cope with daily stress and prevent or slow the onset of aging. While counting his last days on earth, the older one requires to rejuvenate himself. This journey with the requirements mentioned is incomplete without herbal remedies, which supply nutrition and essential ingredients at every step of life to maintain physic [12].

The availability of herbal remedies surpassed drug stores and entered food stores and supermarkets. About 80% of the world’s population, living in the developing world, relies on herbal medicinal products as primary healthcare [9, 13, 14]. With this surge of growing use of herbal medicinal products, a whopping number of herbal preparations are incoming and concern related to safety is surging. A notable share of herbal medicine is used as antidiabetic herbal medicine (ADHM)- due to the number of people suffering from diabetes-related complications crossing 200 million worldwide [15, 16].

Metals are widely distributed in nature and occur freely in soil and water. When the metal has a relatively high density and is toxic at low concentration, it is inked as heavy metal. Among the heavy metals, mercury, lead, arsenic and cadmium are toxic metals and have mutagenic effects even at a very low concentration. Mercury was used to treat syphilis before the introduction of penicillin. Another heavy metal, arsenic, is used for the treatment of some forms of malignancy in the compound form [17, 18]. Therefore, the presence of toxic metals as a physical contaminant in herbal remedies is likely. Metal toxicity may lead to malfunction and malformation of organs. Lead poisoning may cause abdominal pain, vomiting, severe anemia, hemoglobulinuria with dark color stools [17, 18].

A wide spectrum of microorganisms has made their adobe in medicinal plants. A series of influences from animal and inanimate sources is behind this hosting. Bacterial endospore and fungal spores are prime microbial loads associated with herbal plants. These varieties of microbial load are transferred to herbal preparations. Intrinsic and extrinsic factors determine the microbial load of medicinal plants. Certain microbial contaminants may cause severe damage to human health. Certain fungal genera produce mycotoxin, which is a potential health hazard chemical. Ingestion of adherent fungal flora with herbal drugs is associated with human disorders. Not only the microbes but also the low molecular weight metabolites from molds are known as chemical contaminants. Improper handling during production and packaging may give access to microbial load to be into herbal drugs. When plastics, glass and other packaging materials come in contact with medicinal herbs, contamination takes place.

While some herbal medicines have proved potential, many of them remain un-assessed in terms of their safety and efficacy [19]. The absence of proper quality controls, improper labeling and inappropriate patient information are behind the compromised quality of herbal drugs [20]. Herbal drugs are introduced as foods or dietary supplements in some countries. By doing so, the quality, efficacy, and safety of these herbal medicines are not required to comply with drug safety regulations. If tested, then quality tests and production standards are less rigorous or controlled. Not only this, the practitioners who are prescribing the health products, may not be certified or licensed. This would leave the safety of the general public on the verge of decaying [21]. The unlicensed herbal remedy is the commonest route which does not have to meet specific standards of safety and quality, neither is it required to be accompanied by safety information for the consumer [20]. Bangladesh is one of the most populous countries, positioning eighth in the world (Fig. 1). (https://www.infoplease.com/world/population-statistics/worlds-50-most-populous-countries-2016). With small territory, this huge population has made Bangladesh one of the most densely populated in the world.

Unsurprisingly, for a high population with limited wealth, herbal medicines are widely used as medication in Bangladesh. Therefore, the need to educate the physician as well as the general public with adequate information regarding the risks associated with the use of herbal medicines is in demand. With this understanding, a safety investigation of some antidiabetic herbal medicines (ADHMs) in terms of toxic metals (physical contaminants) and microbes (biological contaminants) was taken as our current study.

Fig. (1)) World Population Distribution.

Methods

Study Area and Sample Collection

Samples of eight antidiabetic herbal medicines (ADHMs) as finished commercial packs were purchased randomly from different herbal medicine outlets of Dhaka City. Initially, all the samples were prepared for analysis in the research laboratory of Daffodil International University (DIU), Dhaka, Bangladesh. Microbiological contamination and heavy metal content were analyzed in the Center for Advanced Research in Sciences (CARS), University of Dhaka, Bangladesh.

Determination of pH

The pH of different herbal medicines was determined by using a microprocessor pH meter (HI 2210; Hanna Instrument, USA) [17]. For pH determination, the sample solution was prepared by dissolving 12.5 g in 100 mL sterile distilled water with shaking to obtain a homogeneous solution. By means of a microprocessor pH meter, the solution pH of the different herbal medicines was measured and the data were presented as the average of triplicate.

Microbiological Analysis

Preparation of Culture Media

All the media for microbiological analysis were prepared according to the guidelines of manufacturers’ and sterilized either in an autoclave (CL-32S; ALP Co. Ltd, Japan) at 121oC and 15 psi pressure for 40 minutes, or by heating in a microwave oven when necessary. The sterile media were dispensed or poured into the sterilized Petri dishes or test tubes as required. The sterility of the prepared media was confirmed by incubating blindly selected plates at 37 oC overnight.

Isolation and Identification of Microorganisms

Isolation of microorganisms was done by following standard surface plate technique on various selective and non-selective agar media. For microbial isolation, samples (25 g, when tablets; 25 mL, when syrup) were homogenized with 225 mL autoclaved normal saline (0.85% NaCl). Same amounts of samples were blended separately into different types of pre-enrichment or enrichment broths (225 mL) in case of presence or absence tests for pathogenic microorganisms.

Total Aerobic Bacterial Count and Total Coliform Count

Sample pH was controlled within the range of 6.9 - 7.9 by adding NaOH or HCl when necessary. For microbial isolation, the samples were serially diluted, and with appropriate dilution, 0.1 mL sample was surface plated on a Tryptic Soy agar (TSA; Oxoid Ltd, Hampshire, England) medium and incubated at 35 oC for 24 hrs. To assess the hygiene of the drug formulations, the total coliform count (TCC) was analyzed by following the surface plating technique with 0.1 mL of the sample (as used for TABC) on MacConkey agar (Oxoid, Hampshire, England) and was incubated at 37oC for 24 hrs. All the samples were separately inoculated into the Tryptic Soya broth (Oxoid, England) as a common enrichment medium and incubated at 37 oC for 24 hrs. The broth culture was then streaked onto a MacConkey agar medium and incubated at 37 oC for 24 hrs. The bacterial growth was monitored for further confirmation of the presence or absence of any coliform bacteria in the samples.

Escherichia coli 0157

All the samples were homogenized in the EC medium and incubated at 35oC for 24 hrs. The enriched cultures were streaked onto a Sorbitol MacConkey agar supplemented with Cefixime and potassium tellurite and incubated at 37oC for 24 hrs. For further confirmation of the identification of presumptive colonies, biochemical tests (IMViC) were performed following the standard procedures.

Escherichia coli

All the samples were homogenized in an Enterobacteria enrichment broth-Mossel pre-enrichment medium and incubated at 35 oC for 24 hrs. 1.0 mL aliquots of pre-enriched cultures were mixed with 9 mL of the double strength EC medium and incubated at 37 oC for 24 hrs. One loop full of the culture was inoculated into a 10 mL 1x EC medium with the Durham fermentation tubes and incubated at 42 oC for 24 hrs. Gas production in the Durham tubes was monitored to confirm the presence or absence of E. coli into the 1x EC medium.

Salmonella spp.

All the samples were homogenized independently into the buffered peptone water, followed by incubation at 35 oC for 18-20 hrs. 1.0 mL of pre-enrichment culture was mixed with 9.0 mL of both Hanja Tetrathionate and Rappaport-Vassiliadis broth and incubated for 24 hrs at 35oC and 42 oC, respectively. The culture broths were subsequently streaked onto a Bismuth sulfite agar (BSA; Oxoid, England) and incubated at 37 oC for 24 hrs.

Listeria spp.

Both the tablet and syrup samples were independently supplemented with buffered peptone water and incubated at 30 oC for 4 hrs. 10 mL of broth was then transferred to 90 mL of Listeria enrichment broth and the incubation was continued for further 24 hrs at 30 oC. Finally, one loop full of culture broth was streaked onto a PALCAM Listeria Selective agar (Oxoid, England), which was supplemented with PALCAM selective supplement, and the inoculated culture medium plates were incubated at 35 oC for 24-48 hrs.

Yeasts and Molds Count (YMC)

Total yeasts and mold analyses were performed by following the surface plating technique on the Saboraud’s Dextrose agar (AFC chemical, Bangladesh) with the appropriate dilutions of samples, which were homogenized with sterile normal saline (0.85% NaCl). After 5 days of inoculation at 25o C, the agar plates were observed for the growth of yeasts and molds.

Toxic Metal Analysis

Tablet and capsule samples of antidiabetic herbal medicines (ADHMs) were ground into a fine powder using a mortar and a pestle. Then the powdered samples were digested by taking 3g of each ADHM sample into a separate 100 mL quick fitted round bottom flasks (Pyrex, Germany); 50 mL of 69.5% (w/w) HNO3 was added to each of the flasks and heated until about 10 mL of each of the solution remained, followed by the addition of 2 mL of 60% HClO4 acid, 10 mL of 69.5% (w/w) HNO3 and 1mL of 98% (w/w) H2SO4. The mixtures were further heated in a fume cupboard until the appearance of white fumes. The resulting solutions after cooling were filtered into separate 100 mL volumetric flasks and then diluted to the mark with de-ionized water [22