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“Standardization of a Unani Medicine As well its Raw Materials”
A Dissertation submitted on the thesis work as a requirement for the M.S. Degree in
The Department of Chemistry, University of Dhaka.
Submitted by
Examination Roll No-3902
Registration No:Ha-1539
Session: 2008-2009
October, 2011
Organic Research Laboratory
Department of Chemistry
University of Dhaka
Dhaka, Bangladesh
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Contents Chapter one (Introduction) Page No. 1.1 Introduction-----------------------------------------------------------------------------------------------------8 1.2 Current Status-------------------------------------------------------------------------------------------------10 1.3 Protocols for standardization of herbal drugs---------------------------------------------------------12 1.4 Introduction to sample--------------------------------------------------------------------------------------13 1.4.1 Introduction to Constituents--------------------------------------------------------------------------14 1.4.1.1 Bael Pulp---------------------------------------------------------------------------------------------------14 1.4.1.2 Kurchi Bark------------------------------------------------------------------------------------------------14 1.5 Review Studies on Aegle marmelos----------------------------------------------------------------------15 1.5.1 Chemical Constituents----------------------------------------------------------------------------------16 1.5.2 Traditional Uses of Bael Tree Parts for Medicinal Purpose------------------------------------16 1.6 Review Studies on Holarrhena anti-dysenteria--------------------------------------------------------16 1.6.1 Chemical Constituents--------------------------------------------------------------------------------- 17 1.6.2 Traditional Uses of Kurchi Bark for Medicinal Purpose-----------------------------------------18 1.7 Objective of the present research------------------------------------------------------------------------19
Chapter two (Methodology)
2.1.1 Solvents and reagents-------------------------------------------------------------------------------------20
2.1.2 Distillation of the solvents--------------------------------------------------------------------------------20 2.1.3. Evaporation-------------------------------------------------------------------------------------------------20 2.1.4. Freeze-drying---------------------------------------------------------------------------------------------21 2.2 Preparation of extracts--------------------------------------------------------------------------------------21 2.2.1 Initial extraction by Decoction Method---------------------------------------------------------------21 2.2.1.1 Extraction of BAEL PULP (Aegle marmelos)-------------------------------------------------------21 2.2.1.2 Extraction of KURCHI BARK (Holarrhena anti-dysenteria)-------------------------------------22 2.2.1.3 Evaporation-----------------------------------------------------------------------------------------------22 2.2.1.4 Freeze Drying---------------------------------------------------------------------------------------------------------23 2.3 Chromatographic techniques------------------------------------------------------------------------------23 2.3.1 Thin layer chromatography (TLC)-----------------------------------------------------------------------23 2.3.2 Sample application (spotting the plates)-------------------------------------------------------------24 2.3.3 Preparation of TLC tank----------------------------------------------------------------------------------24 2.3.4 Solvent Systems-------------------------------------------------------------------------------------------25 2.3.5 Visualization/detection of compounds--------------------------------------------------------------25 2.3.6 Vanillin-sulphuric acid reagent------------------------------------------------------------------------25 2.3.7 Determination of Rf (Retention factor) values-----------------------------------------------------25 2.3.8 Physiochemical Screening------------------------------------------------------------------------------26 2.3.8.1 Test for Alkaloids---------------------------------------------------------------------------------------26 2.3.8.1.1 Preparation of Mayer’s reagent------------------------------------------------------------------26 2.3.8 Physiochemical Screening------------------------------------------------------------------------------27 2.3.8.1 Test for Alkaloids---------------------------------------------------------------------------------------27
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2.3.8.1.1 Preparation of Mayer’s reagent--------------------------------------------------------------------------27 2.3.8.2 Test for Cardiac glycoside------------------------------------------------------------------------------------27 2.3.8.3 Test for Terpenoids--------------------------------------------------------------------------------------------27 2.3.8.4 Test for Saponins-----------------------------------------------------------------------------------------------27 2.3.8.5 Test for Tannins------------------------------------------------------------------------------------------------27 2.3.8.6 Test for Flavonoids--------------------------------------------------------------------------------------------27 2.3.8.7 Test for Steroids------------------------------------------------------------------------------------------------27 2.3.8.8 Test for Phlobatannins---------------------------------------------------------------------------------------28 2.4 Spectroscopic techniques----------------------------------------------------------------------------------------28 2.4.1 Infra-red (IR) spectroscopy------------------------------------------------------------------------------------28 2.4.1.1 Sample Preparation-------------------------------------------------------------------------------------------29 2.7.2 Ultra-violet (UV) spectroscopy-------------------------------------------------------------------------------29 2.5 Ash Content Analysis----------------------------------------------------------------------------------------------30 2.5.1 Flame Photometer----------------------------------------------------------------------------------------------31 2.5.1.1 Determination of Sodium (Na) Content by Flame Photometer----------------------------------31 2.5.1.2 Determination of Potassium (K) Content by Flame Photometer--------------------------------31 2.5.2 Determination of Heavy Metals Using Atomic Absorption Spectroscopy (AAS)-----------------32 2.5.2.1 Atomic Absorption Spectroscopy (AAS)-----------------------------------------------------------------32 2.5.2.2 Determination of Palladium (Pd), Copper (Cu),Cadmium (Cd) & Chromium ( Cr)------------33 2.5.2.3 Determination of Manganese (Mn), Cobalt (Co), Nickel (Ni) & Zinc (Zn)-----------------------33 2.5.2.4 Determination of Calcium (Ca)----------------------------------------------------------------------------33 2.5.2.4 Determination of Arsenic (As) & Mercury (Hg)--------------------------------------------------------34 Chapter Three (Experimental) 3.1 Collection and preparation of pulp of Aegle marmelos-------------------------------------------------------36 3.2 Collection and preparation of bark of Holarrhena anti-dysenteria-----------------------------------------36 3.3 Collection of Commercial samples---------------------------------------------------------------------------------36 3.4 Determination of Foreign matter--------------------------------------------------------------------------37 3.4.1 Procedure-----------------------------------------------------------------------------------------------------37 3.4.2 Experimental data------------------------------------------------------------------------------------------37 3.5 Determination of Moisture Content---------------------------------------------------------------------37 3.5.1 Procedure---------------------------------------------------------------------------------------------------37 3.5.2 Experimental data----------------------------------------------------------------------------------------38 3.6 Determination of Ash content--------------------------------------------------------------------------------------39 3.6.1 Procedure--------------------------------------------------------------------------------------------------------------39 3.6.2 Experimental data-------------------------------------------------------------------------------------------39 3.7 Determination of Extractable Matter (Decoctions method)----------------------------------------41 3.7.1 Extraction Procedure of Bael Pulp (Aegle marmelos)-------------------------------------------------------41 3.7.2 Experimental data----------------------------------------------------------------------------------------------------42 3.7.3 Extraction Procedure of Kurchi Bark (Holarrhena anti-dysenteria)--------------------------------------42 3.7.4 Experimental data----------------------------------------------------------------------------------------------------43
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Chapter four (Ash Content Analysis) 4.1 Determination of Sodium (Na) content by Flame Photometer------------------------------------------44 4.1.1 Apparatus---------------------------------------------------------------------------------------------------------44 4.1.2 Solution Preparation-------------------------------------------------------------------------------------------44 4.1.3 Experimental Data----------------------------------------------------------------------------------------------44 4.1.4 Calibration Curve-----------------------------------------------------------------------------------------------45 4.2 Determination of Potassium (K) content by Flame Photometer----------------------------------------46 4.2.1 Apparatus--------------------------------------------------------------------------------------------------------46 4.2.2 Solution Preparation------------------------------------------------------------------------------------------46 4.2.3 Experimental Data---------------------------------------------------------------------------------------------46 4.2.4 Calculation-------------------------------------------------------------------------------------------------------46 4.2.5 Calibration Curve-------------------------------------------------------------------------------------------------47 4.3 Determination of Heavy Metals by Atomic Absorption Spectrometry (AAS) -----------------------47 4.3.1 Determination of Palladium (Pd), Copper (Cu),Cadmium (Cd) & Chromium ( Cr)----------------47 4.3.1.1 Apparatus--------------------------------------------------------------------------------------------------------47 4.3.1.2 Solution Preparation------------------------------------------------------------------------------------------48 4.3.1.3 Experimental Data---------------------------------------------------------------------------------------------48 4.3.2 Determination of Manganese (Mn), Cobalt (Co), Nickel (Ni) & Zinc (Zn)---------------------------49 4.3.2.1 Apparatus--------------------------------------------------------------------------------------------------------49 Machine Details----------------------------------------------------------------------------------------------------------49 4.3.2.2 Solution Preparation------------------------------------------------------------------------------------------49 4.3.2.3 Experimental Data---------------------------------------------------------------------------------------------49 4.3.3 Determination of Calcium (Ca)-------------------------------------------------------------------------------50 4.3.3.1 Apparatus--------------------------------------------------------------------------------------------------------50 4.3.3.2 Solution Preparation------------------------------------------------------------------------------------------50 4.3.3.3 Flame/calibration Standard measurement--------------------------------------------------------------51 4.3.3.4 Calibration Curve----------------------------------------------------------------------------------------------52 4.3.3.5 Experimental Data----------------------------------------------------------------------------------------------52 4.3.4 Determination of Arsenic (As)--------------------------------------------------------------------------------53 4.3.4.1 Apparatus------------------------------------------------------------------------------------------------------53 4.3.4.2 Solution Preparation-----------------------------------------------------------------------------------------54 4.3.4.3 Reagents---------------------------------------------------------------------------------------------------------54 4.3.4.4 Procedure--------------------------------------------------------------------------------------------------------54 4.3.4.5 Flame/calibration Standard measurement--------------------------------------------------------------55 4.3.4.6 Calibration Curve------------------------------------------------------------------------------------------------55 4.3.4.7 Experimental Data----------------------------------------------------------------------------------------------55 4.3.5 Determination of Mercury (Hg)-------------------------------------------------------------------------------56 4.3.5.1 Apparatus--------------------------------------------------------------------------------------------------------56 4.3.5.2 Solution Preparation------------------------------------------------------------------------------------------56 4.3.5.3 Reagents---------------------------------------------------------------------------------------------------------56 4.3.5.4 Procedure-------------------------------------------------------------------------------------------------------56 4.3.5.5 Flame/calibration Standard measurement--------------------------------------------------------------57
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4.3.5.6 Calibration Curve-----------------------------------------------------------------------------------------------57 4.3.5.7 Experimental Data---------------------------------------------------------------------------------------------57 Chapter Five (Extractable Matter Analysis) 5.1 Thin layer chromatography of the Freeze Dried Extract & Commercial Samples---------------------58 5.1.1 Development and determination of the Solvent System-------------------------------------------------58 5.1.2 Procedure------------------------------------------------------------------------------------------------------------59 5.2 Spectroscopic analysis of the Freeze Dried Extract & Commercial Samples---------------------------60 5.2.1 Infrared Spectroscopy--------------------------------------------------------------------------------------------60 5.2.1.1 IR Spectra of Bael Pulp (Aegle marmelos)-----------------------------------------------------------------61 5.2.1.1.1 Spectroscopic characteristics---------------------------------------------------------------------------61 5.2.1.2 IR Spectra of Kurchi Bark (Holarrhena anti-dysenteria)------------------------------------------------62 5.2.1.2.1 Spectroscopic characteristics---------------------------------------------------------------------------62 5.2.1.3 IR Spectra of Bel Syrup (Ibne Sina)--------------------------------------------------------------------------63 5.2.1.3.1 Spectroscopic characteristics---------------------------------------------------------------------------63 5.2.1.4 IR Spectra of Syrup anti-dysenteria (New Life)----------------------------------------------------------64 5.2.1.4.1 Spectroscopic characteristics---------------------------------------------------------------------------64 5.2.1.5 IR Spectra of Syrup Marbelus (Hamdard)-----------------------------------------------------------------65 5.2.1.3.1 Spectroscopic characteristics---------------------------------------------------------------------------65 5.2.2 UV Spectroscopy---------------------------------------------------------------------------------------------------66 5.2.2.1 UV Spectra of Bael Pulp (Aegle marmelos)---------------------------------------------------------------67 5.2.2.2 UV Spectra of Kurchi Bark (Holarrhena anti-dysenteria)----------------------------------------------68 5.2.2.3 UV Spectra of Bel Syrup (Ibne Sina)------------------------------------------------------------------------69 5.2.2.4 UV Spectra of Syrup anti-dysenteria (New Life)---------------------------------------------------------70 5.2.2.5 UV Spectra of Syrup Marbelus (Hamdard)----------------------------------------------------------------71 5.3 Phytochemical Screening of Raw Materials & Commercial Samples------------------------------------72 5.3.1 Phytochemical Screening of Bael Pulp (Aegle marmelos)------------------------------------------------72 5.3.1.1 Test for Alkaloids------------------------------------------------------------------------------------------------72 5.3.1.1.1 Preparation of Mayer’s reagent---------------------------------------------------------------------------72 5.3.1.2 Test for Cardiac glycoside-------------------------------------------------------------------------------------72 5.3.1.3 Test for Terpenoids---------------------------------------------------------------------------------------------72 5.3.1.4 Test for Saponins------------------------------------------------------------------------------------------------72 5.3.1.5 Test for Tannins--------------------------------------------------------------------------------------------------73 5.3.1.6 Test for Flavonoids----------------------------------------------------------------------------------------------73 5.3.1.7 Test for Steroids-------------------------------------------------------------------------------------------------73 5.3.1.8 Test for Phlobatannins----------------------------------------------------------------------------------------73 5.3.2 Phytochemical Screening of Kurchi bark--------------------------------------------------------------------74 5.3.1.1 Test for Alkaloids-----------------------------------------------------------------------------------------------74 5.3.1.1.1 Preparation of Mayer’s reagent--------------------------------------------------------------------------74 5.3.1.2 Test for Cardiac glycoside------------------------------------------------------------------------------------74 5.3.1.3 Test for Terpenoids--------------------------------------------------------------------------------------------74 5.3.1.4 Test for Saponins-----------------------------------------------------------------------------------------------74 5.3.1.5 Test for Tannins-------------------------------------------------------------------------------------------------75
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5.3.1.6 Test for Flavonoids---------------------------------------------------------------------------------------------75 5.3.1.7 Test for Steroids------------------------------------------------------------------------------------------------75 5.3.1.8 Test for Phlobatannins----------------------------------------------------------------------------------------75 Chapter Six (Results & Discussion) 6.1 Heavy Metal Analysis----------------------------------------------------------------------------------------------78 6.2 Extractable Metal Analysis---------------------------------------------------------------------------------------80 6.2.1 Thin layer Chromatography Analysis------------------------------------------------------------------------80 6.2.2 IR Spectroscopic Analysis--------------------------------------------------------------------------------------81 6.2.2.1 IR & UV spectroscopic analysis of bael Pulp (Aegle marmelos)------------------------------------81 6.2.2.2 IR & UV spectroscopic analysis of Kurchi Bark (Holarrhena anti-dysenteria)-------------------81 6.2.2.3 IR & UV spectroscopic analysis of Bel Syrup (Ibne Sina)---------------------------------------------81 6.2.2.4 IR & UV spectroscopic analysis of Syrup anti-dysenteria (New life)------------------------------81 6.2.2.5 IR & UV spectroscopic analysis of Syrup Marbelus (Hamdard)------------------------------------81 6.3 Conclusion-----------------------------------------------------------------------------------------------------------83 References---------------------------------------------------------------------------------------------------------------84 ACRONYMS--------------------------------------------------------------------------------------------------------------86
List of figures
Fig 1.1 : Leaves and fruits of Bael-----------------------------------------------------------------------------------14
Fig 1.2 : Kurchi Bark plant----------------------------------------------------------------------------------------16
Fig 2.1 : Rotary Evaporator (BUCHI)---------------------------------------------------------------------------19
Fig 2.2 : Powdered Kurchi Bark (Holarrhena anti-dysenteria)------------------------------------------20
Fig 2.3 : Freeze Dried extract of Bael Pulp (Aegle marmelos)------------------------------------------21
Fig 2.4 : Freeze Dried extract of KURCHI BARK (Holarrhena anti-dysenteria)---------------------22
Fig 2.5 : Spotting of TLC plate----------------------------------------------------------------------------------23
Fig 2.5 : Spotting of TLC plate----------------------------------------------------------------------------------24
Figure 2.7: A Plate for the calculation of R f value--------------------------------------------------------25
Fig 2.8 : Schematics of a two-beam absorption spectrometer---------------------------------------27
Fig 2.9 : Shimadzu IR-470 spectrometer--------------------------------------------------------------------27
Fig 2.10 : Diagram of a single-beam UV/Vis spectrophotometer--------------------------------------28
Fig 2.11: Shimadzu UV-160A spectrometer----------------------------------------------------------------29
Fig 2.12 : Muffle Furnace(Barnstead Thermolyne, Model-48000)------------------------------------29
Fig 2.13 : Flame Photometer (JENWAY,Model = PFP7 Flame Photometer)------------------------30
Fig 2.14 : Atomic Absorption Spectrometer block diagram--------------------------------------------31
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Fig 2.15 : Atomic Absorption Spectrometer (Varian ,Model = AA240FS-Fast Sequential Atomic Absorption)—32
Fig 2.16 : Atomic Absorption Spectrophotometer (Shimadzu Model =AA6401F)-------------------33
Fig 2.17 : AAS with VGA--------------------------------------------------------------------------------------------------------33
Fig 2.18 : Vapor Generation Accessories (Model VGA-77)-------------------------------------------------33
Fig 3.1 : Commercial samples-------------------------------------------------------------------------------------34
Fig 3.2 : Ash of raw materials-------------------------------------------------------------------------------------37
Fig 3.3 : Ash of commercial samples--------------------------------------------------------------------39
Fig 3.4 : Extractable Matter of Bael Pulp (Aegle marmelos)----------------------------------------------41
Fig 3.5 : Extractable Matter of Kurchi Bark (Holarrhena anti-dysenteria)-----------------------------42
Fig 4.1 : Flame Photometer (JENWAY,Model = PFP7 Flame Photometer)----------------------------43
Fig 4.2: Calibration curve for Sodium (Na)-------------------------------------------------------------------44
Fig 4.3: Calibration curve for Potassium (K)-----------------------------------------------------------------46
Fig 4.6 : Calibration Curve for Manganese (Mn), Cobalt (Co), Nickel (Ni) & Zinc (Zn)------------51
Fig 4.9 : Quartz Absorption cell for Arsenic Determination-----------------------------------------------52
Fig 4.10 : Solutions used for determining Arsenic Content-----------------------------------------------53
Fig 4.11 : Calibration Curve for Arsenic (As)-----------------------------------------------------------------54
Fig 4.12 : Calibration Curve for Mercury (Hg)---------------------------------------------------------------56
Fig 5.1 : Thin layer chromatography of the Freeze Dried Extract & Commercial Samples-------57
Fig 5.2: TLC chromatogram of raw & commercial samples---------------------------------------------58
Fig 5.4 : IR Spectra of Bael Pulp (Aegle marmelos)-------------------------------------------------------60
Fig 5.5 : IR Spectra of Kurchi Bark (Holarrhena anti-dysenteria)-------------------------------------61
Fig 5.6 : IR Spectra of Bel Syrup (Ibne Sina)---------------------------------------------------------------62
Fig 5.7 : IR Spectra of Syrup anti-dysenteria (New Life)-----------------------------------------------63
Fig 5.8 : IR Spectra of Syrup Marbelus (Hamdard)------------------------------------------------------64
Fig 5.10 : UV Spectra of Bael Pulp (Aegle marmelos)--------------------------------------------------66
Fig 5.11 : UV Spectra of Kurchi Bark (Holarrhena anti-dysenteria)---------------------------------67
Fig 5.12 : UV Spectra of Bel Syrup (Ibne Sina)----------------------------------------------------------68
Fig 5.13 : UV Spectra of Syrup anti-dysenteria (New Life)------------------------------------------69
Fig 5.14 : UV Spectra of Syrup Marbelus (Hamdard)-------------------------------------------------70
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Introduction
Traditional Unani system of medicine implies knowledge and practice of herbal healing for the
prevention, diagnosis and elimination of physical, mental, or social imbalance. The costs for health care
are rising at an alarming rate throughout the world. At the same time, the world market for
phytopharmaceuticals is growing steadily. The World Bank estimates that trade in medicinal plants,
botanical drug products, and raw materials are growing at an annual rate of between 5 and 15 % [1–3].
The proportion of plant-derived drugs differs from country to country. For example, while
approximately 25 % of prescriptions in the United States contained plant-derived active ingredients,
40% of the drugs on the German list of medicines (“Rote List”) were based on plant material [4]. In
some countries such as Malaysia, the plant origins of routinely prescribed medicines are easily
accessed and well documented. Herbs, used as medicine, are also regulated under different categories
throughout the world. Institutionally prepared formulas are often readily available without
prescription. In some European countries, standardized concentrated extracts are regulated as drugs
which can be obtained by prescription only. Herbs are considered to be dietary supplements in the
United States and therefore are subjected to a very limited form of regulation and oversight [5].
It is a common observation that people diagnosed with incurable chronic disease states such as
diabetes, arthritis, and AIDS turned to herbal therapies for a sense of control and mental comfort from
taking action [54]. Herbal product studies cannot be considered scientifically valid if the product tested
has not been authenticated and characterized in order to ensure reproducibility in the manufacturing
of the product in question. Several studies have indicated quantitative variations in marker
constituents in herbal preparations. Moreover, many dangerous and lethal side effects have recently
been reported, including direct toxic effects, allergic reactions, effects from contaminants, and
interactions with drugs and other herbs. Of the 10 most commonly used herbs in the United States,
systematic reviews have concluded that only 4 are likely to be effective and there is very limited
evidence to evaluate the efficacy of the approximately 20 000 other available herbal products [6].
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Recent surveys reported in the American news media indicated that a large percentage of the public
would like to see products supported by science, which means products supported by clinical &
laboratory research. This means consumers are increasingly demanding products of known quality.
This is where the public standards enter into the picture [7]. There is a strong demand and need to
accelerate the research in phytomedicine [8].
Standardized herbal products of consistent quality and containing well-defined constituents are
required for reliable clinical trials and to provide consistent beneficial therapeutic effects.
Pharmacological properties of an herbal formulation depend on phytochemical constituents present
therein. Development of authentic analytical methods which can reliably profile the phytochemical
composition. Without consistent quality of a phytochemical mixture, a consistent pharmacological
effect is not expected. Resurgence of interest and the growing market of herbal medicinal products
necessitate strong commitment by the stakeholders to safeguard the consumer and the industry.
Standardization is the first step for the establishment of a consistent biological activity, a consistent
chemical profile, or simply a quality assurance program for production and manufacturing.
Therefore, the EU has defined three categories of herbal products:
• those containing constituents (single compounds or families of compounds) with known and
experienced therapeutic activity that are deemed solely responsible for clinical efficacy;
• those containing chemically defined constituents possessing relevant pharmacological properties
which are likely to contribute to the clinical efficacy; and
• those in which no constituents have been identified as being responsible for the therapeutic activity.
Standardization as defined in the text for guidance on the quality of herbal medicinal products means
adjusting the herbal drug preparation to a defined content of a constituent or group of substances with
known therapeutic activity. The European Medicines Agency (EMEA) makes the distinction between
constituents with known therapeutic activity which can be used to standardize a biological effect and
marker compounds which allow standardization on a set amount of the chosen compound.
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1.2 Current Status
Herbs are generally defined as any form of a plant or plant product, including leaves, stems, flowers,
roots, and seeds [64]. Herbal products may contain a single herb or combinations of several different
herbs that are believed to have complementary effects. Some herbal products, including those of TCM
formulations, also include animal products and minerals [9]. Herbal products are sold as either raw
plants or extracts of portions of the plant. The extraction involves boiling, percolating, or macerating
the herb in water, ethanol, or other solvents to release biologically active constituents from the cell
matrices of the plant into the solvents. The herb to be extracted may be in its dried or fresh forms.
Regulatory requirements for the quality of herbal products vary depending on the country and the
regulatory category. The same herbal product can be marketed as a drug in Europe and as a dietary
supplement in the United States. In Europe, medicinal plant products are produced according to
quality standards typical for pharmaceutical products. This is especially true for potent herbal products
in which the active ingredients are defined, contribute substantially to the therapeutic activity, and
allow standardization of a constituent(s) within a set range supported by a pharmacopoeial
monograph.
Individual governments, the WHO, and panels of academic experts and clinicians often provide
guidelines for manufacturing and quality control, as well as therapeutic use in terms of indication,
dose, side effects, and possible safety concerns. Many of these guidelines are compiled in
pharmacopoeial monographs. These guidelines are governed by regulations that cover all aspects from
manufacturing to labeling and advertising of the finished products. In the United States, compliance of
dietary supplements to a pharmacopoieal monograph is optional. Thus, it is difficult for consumers of
dietary supplements to make informed decisions about self-medication based upon label information.
The level of quality control employed by different manufacturers varies widely. Claims of
standardization are made without definition of the term or indication of whether the chemicals used in
standardization are responsible for therapeutic effect. Without all this information, the consumers can
make purchasing decisions based upon price only.
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Examples of Monographs
• American Herbal Pharmacopoeia: 4 monographs (type: standards, therapeutic; includes Chinese and
Ayurvedic herbs).
• British Herbal Pharmacopoeia: 169 monographs (type: standards; referenced, revised in 1996)
• British Herbal Compendium: 84 monographs corresponding to 84 herbs listed in 1990 edition of BHP
(type: therapeutic; referenced)
• European Scientific Cooperation for Phytotherapy (ESCOP): 50 herbal monographs (type: therapeutic;
referenced)
• German Commission E (ABC translation): 433 monographs (including revisions),324 herb and
combinations, 200 approved herb (type: therapeutic; no longer being evaluated and produced by
German government; not referenced) [10].
• United States Pharmacopoeia: 11 monographs (type: standards (8), therapeutic (3); therapeutic
monographs)
• World Health Organization: 28 monographs covering 31 plant species (type: standards, therapeutic)
The most important step in the development of analytical methods for botanicals and herbal
preparations is sample preparation. The basic operation includes steps such as pre-washing, drying of
plant materials, or freeze-drying and grinding, to obtain a homogenous sample and often improving
the kinetics of extraction of the constituents. In the pharmacopoeial monographs, methods such as
sonication, heating under reflux, Soxhlet extraction, and others are commonly used [11,12].
Separation of individual components from the herbal mixture is the key step to enable identification
and bioactivity evaluation. Chromatography is a powerful analytical method suitable for the separation
and quantitative determination of a considerable number of compounds, even from a complex
matrix. These include paper chromatography (PC), thin-layer chromatography (TLC), gas
chromatography (GC), HPLC, and capillary electrophoresis (CE). UV absorption has been the most
commonly used detection method for the preliminary identification of the separated components.
However, various other detectors, such as fluorescence (FD), flame ionization (FID), electron capture
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(ECD), refractive index (RI), and most recently, evaporative light scattering (ELSD), are also available for
specific cases.
The presence of toxic metals is also one of the parameters included in pharmacopoeias. The tool
primarily used to detect and quantify the elements in most analyses is based on atomic absorption
spectrometry (AAS).
1.3 Protocols for Standardization of Herbal Drugs
In order to assure a consistent and acceptable quality herbal product, care should be taken right from
the identification and authentication of herbal raw materials to the verification process of final
product.
The following parameters are recommended.
1. Authentication The first stage is identification of the plant species or
botanical verification by the currently accepted Latin
binomial name and synonyms [13]. The steps involved in
authentication are taxonomic, and macroscopic and
microscopic studies. Records should be maintained for stage
of collection, parts of the plant collected, regional status,
botanical identity such as phytomorphology, microscopial,
and histological analysis, taxonomical identity, etc.
2. Physical parameters Physical tests [14] include organoleptic evaluation (sensory
characters such as taste, appearance, odor, feel of the drug,
etc.), viscosity, moisture content, pH, disintegration time,
friability, hardness, flow ability, sedimentation, and ash
value.
3. Chromatographic and
spectroscopic evaluation
Sophisticated modern techniques of standardization such as
UV–vis spectrophotometry, TLC, HPTLC [14,15], HPLC
[16-19], NMR [20,21], near infrared spectroscopy [94]
provide quantitative and semi quantitative information
about the main active constituents or marker compounds
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present in the crude drug or herbal products.
Markers play an important role in fingerprinting of herbs.
Quality of drug can also be assessed by chromatographic
fingerprint [22].
4. Microbiological parameters Microbiological contamination can be measured according to
methods described in the Romanian Pharmacopoeia [22], as
well as in the British Pharmacopoeia [23]. Microbiological
analysis includes analysis of limits of E. coli and molds, total
viable aerobic count, total enteriobacteria and their count,
aflatoxin analysis.
5. Pesticide residue analysis Standard limits of pesticides have been set by WHO and
FAO (Food and Agricultural Organization). Some common
pesticides that cause harm to human beings, such as DDT,
BHC, toxaphene, and aldrin, should be analyzed [24-27].
6. Heavy metal analysis Toxic metals such as Cu, Zn, Mn, Fe, and particularly Cd,
As, Pb and Hg should be analyzed [28-30]. In the analysis
of metals, their speciation is to be taken into consideration
1.4 Introduction to Sample
Generic Name : Bael Giri
Sample Name : Bel Syrup (Ibne Sina)
Syrup anti-dysenteria (New Life)
Syrup Marbelus (Hamdard)
Constituents
1. Bael Pulp
2. Kurchi bark
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1.4.1 Introduction to Constituents
1.4.1.1 Bael Pulp
Local Name : Bael
Scientific Name : Aegle marmelos
Subjective Name : Bengal quince, wood apple, stone apple
Family : Rutaceae (Citrous family)
Used Part : Fibrous Yellow Pulp
Collection date : 23/11/10
Collector’s Name : Md.Abrar Mahir Khan
Identifier Name : Dr. Mostafa
Source : Kunjalal Pitambar Shah
266,Nawabpur Road,
Dhaka-1100
01191106310
1.4.1.1 Kurchi Bark
Local Name : Kurchi bark
Scientific Name : Holarrhena anti-dysenteria
Subjective Name : Kutaj, Bitter Oleander, Connessi Bark, Dysentery Rose Bay
Family : Apocynaceae
Used Part : Bark
Collection date : 23/11/10
Collector’s Name : Md.Abrar Mahir Khan
Identifier Name : Dr. Mostafa
Source : Kunjalal Pitambar Shah
266,Nawabpur Road,
Dhaka-1100
01191106310
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1.5 Review Studies of Aegle marmelos
Bael (Aegle Marmelos (Linn), family Rutacae, is also known as Bale fruit tree, is a moderate sized ,
slender, aromatic tree, 6.0 -7.5 m in height, and 90 to 120 cm in girth, with a somewhat fluted bole of
3.0-4.5 meter growing wild throughout the deciduous forests of Bangladesh. Leaves, fruit, stem and
roots of this tree at all stages of maturity are used as ethno medicine against various human ailments.
Fig 1.1 : Leaves and fruits of Bael
1.5.1 Chemical Constituents: Various phytoconstituents have been isolated from the various parts of
Aegle marmelos, which may be categorized as
Table 1: Phytoconstituents isolated from various parts of Aegle marmelos
Part Phytoconstituents
Leaf Skimmianine, Aegeline, Lupeol, Cineol, Citral, Citronella,
Cuminaldehyde, Eugenol, Marmesinine
Bark Skimmianine, Fagarine , Marmin
Fruit Marmelosin, Luvangetin, Aurapten, Psoralen, Marmelide, Tannin
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1.5.2 Traditional Uses of Bael Tree Parts for Medicinal Purposes
The different parts of Bael are used for various therapeutic purposes, such as for treatment of Asthma,
Anaemia, Fractures, Healing of Wounds, Swollen Joints, High Blood Pressure, Jaundice, Diarrhoea
Healthy Mind and Brain Typhoid Troubles during Pregnancy.
Aegle marmelos has been used as a herbal medicine for the management of diabetes mellitus
in Ayurvedic, Unani and Siddha systems of medicine in India , Bangladesh and SriLanka.
The main usage
of the parts of this tree is for medicinal purposes. The unripe dried fruit is astringent, digestive,
stomachic and used to cure diarrhea and dysentery. Sweet drink prepared from the pulp of fruits
produce a soothing effect on the patients who have just recovered from bacillary dysentery.
The ripe fruit is a good and simple cure for dyspepsia. The pulp of unripe fruit is soaked in gingelly oil
for a week and this oil is smeared over the body before bathing. This oil is said to be useful in removing
the peculiar burning sensation in the soles. The roots and the bark of the tree are used in the
treatment of fever by making a decoction of them. The leaves are made into a poultice and used in the
treatment of opthalmia. The leaf part of the plants have been claimed to be used for the treatment of
inflammation, asthma, hypoglycemia, febrifuge, hepatitis and analgesic. The mucilage of the seed is a
cementing material. The wood takes a fine polish and is used in building houses, constructing carts,
agricultural implements. A yellow dye is obtained from the rind of the unripe fruits. The dried fruits,
after their pulp separated from the rind are used as pill boxes for keeping valuable medicines, sacred
ashes and tobacco. In Homeopathic treatments it is largely used for conjunctivitis and styes, rhinitis,
coccygodynia, nocturnal seminal emission with amorous dreams, chronic dysentery. Ayurveda
prescribes the fruit of the herb for heart, stomach, intestinal tonic, chronic constipation and dysentery;
some forms of indigestion, typhoid, debility, cholera, hemorrhoids, intermittent fever, hypocondria,
melancholia and for heart palpitation. The unripe fruit is medicinally better than the ripe fruit. Leaf
poultice is applied to inflammation; with black pepper for edema, constipation and jaundice.
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1.6 Review Studies of Holarrhena anti-dysenteria
Holarrhenais a deciduous laticiferous shrub or can be considered as a small tree. The tree grows up to
three meters high. The tree has short stem that has pale bark and several branches. The ovate to
obtusely acuminate leaves are 10-20 cm in length. Its leaf stalks are very short.
The Holarrhena has white flowers that appear in corymb-like cymes, 5-15 cm across and present at the
end of the tree’s branches. Kutaja’s flowers of have five white petals 2-3 cm in length that will turn
creamish yellow as they grow. The tree’s flowers have oblong petals that are rounded at the tip.
If more specified, H.antidysenterica is a broad-leafed shrub or small tree. The tree’s bark is rather
rough, pale brownish or grayish and the leaves are opposite, subsessile, elliptic or ovate-oblong,
membranous and the flowers are white, in terminal corymbose cymes; the follicles, divaricate, cylindric
and generally having white spots; the seeds are light brown in color.
Fig 1.2 : Kurchi Bark plant
1.6.1 Chemical Constituents
Various phytoconstituents have been isolated from the various parts of Holarrhena anti-dysenteria,
which may be categorized as
Table 1: Phytoconstituents isolated from various parts of Holarrhena anti-dysenteria
Part Phytoconstituents
Bark Conessine, kurchine, kurchicine, holarrhimine, conarrhimine, conaine,
conessimine, iso-conessimine, conimine, holacetin and conkurchin
18 | P a g e
1.6.2 Traditional Uses of Kurchi Bark for Medicinal Purposes
It is one of the best drug for diarrhoea. In chronic diarrhoea & to check blood coming from stool, it
should be given with Isobgol, castor oil or Indrayav. According to Ayurveda, the bark is useful in
treatment of piles, skin diseases and biliousness.
The bark is used externally in case of skin troubles. The bark is mostly mixed with cow urine and applies
it in affected parts.
In treatment of urinary troubles, the bark is given with cow milk.
The fresh juice of bark is considered good to check the diarrhoea.
Application of this herb is useful in Rh. Arthritis & Osteoarthritis.
The bark is used in chest affections and as a remedy in diseases of the skin and spleen.
It is a well known herb for amoebic dysentry and other gastric disorders.
The bark is used as an astringent, anthelmintic, antidontalgic,stomachic, febrifuge, antidropsical,
diuretic, in piles, colic, dyspepsia, chest affections and as a remedy in diseases of the skin and spleen.
It is a well known drug for amoebic dysentery and other gastric disorders.
It is also indicated in diarrhoea, indigestion, flatulence and colic.
The herb is helpful in augmenting digestion and appetite.
It works well in ano-rectal problems, like proctitis, painful defecation, rectal swellings, etc.Because of
its styptic property, bitter oleander assists in arresting the bleeding piles.
Orally kutaj is effectively used in various maladies; It works well in the treatment of diarrhea and
dysentery, associated with bleeding as well. Since centuries, it has been used as a household remedy
for the same.
It also works well in other ano-rectal problems like proctitis, painful defecation, rectal swellings etc,
Kutaj is beneficial also in skin diseases, especially of oozing type. It can be used as an adjuvant in the
treatment of obesity to get rid of excessive fats.
The herb is also useful in gout as well as raktapitta. The skin of the bark, grated in cow’s milk wirks well
in painful, difficult micturition and in urinary stones also.
Kutaj skin and vidanga seed powder is a popular household remedy for intestinal worm infestations in
children.
19 | P a g e
Kutajarista and Kutajavaleha are the most popular preparations used in diarrhea, dysentery, colitis and
bleeding problems.
1.7 Objective of The Present Research
Over the past decade traditional medicine (botanicals) has become a topic of increasing global
importance, with both medical and economic implications. In developing countries such as Bangladesh,
about 80% of the indigenous populations are dependent on traditional systems of medicine and
medicinal plants as their primary source of healthcare. In the industrialized nations such as the United
States, over 50% of consumers use botanicals as part of complementary and alternative therapies.
Such widespread use of herbals medicines has lead to significant concerns about the quality, safety and
efficacy of these products. Therefore, the aim of the present study is to develop a chemical profile of
these Unani drug as well its basic raw materials to ensure it quality.
Bael Giri (generic name) is a well-known plant drug in Ayurvedic and Unani medicine, which has been
used for the treatment of various diseases and disorders particularly for diarrhea and dysentery. Two
plants named Bael (Aegle marmelos) and Kurchi Bark (Holarrhena anti-dysenteria) is being used for the
formulation of this drug. Chemical profiling is helpful to the identification of the plant species and
improves the quality control of the plant product.
The following steps will be carried out during the present study. Firstly, the bael giri of three different
manufacturer and its raw materials were collected from the local market. Secondly, the raw material
will be dried and grind to powder using an electric grinder. Then the dried powder and finished product
will be extracted with suitable solvents with constant shaking for three times. The extracts will be
screened for their claimed activity by different screening methods. Finally, the Fingerprint analysis of
these formulations as well as its raw materials will be carried out by using TLC. The micronutrient
elements will also be quantified in the different formulation and its raw materials by Flame
Photometry & AAS.
20 | P a g e
METHODOLOGY
2.1.1 Solvents and Reagents
Analytical or laboratory grade solvents and chemicals were used in most of the experiments described
in the thesis that were procured from E. Merck (Germany) and BDH (England). Some commercial grade
solvents were used after distillation in glass distillation set. Solvents used in different experiments
included ethyl- and methyl alcohol, chloroform, n-butanol, acetone, ethyl acetate, dichloromethane, n-
hexane and pet ether etc. Analytical grade of acetic acid, sulphuric acid, vanillin-sulphuric acid mixture,
hydrochloric acid and trifluoro acetic acid (TFA) were also used.
2.1.2 Distillation of the solvents
The commercial grade solvents (dichloromethane, ethyl acetate, chloroform and methanol) were
distilled. Distilled solvents were used through the investigation.
2.1.3. Evaporation
All types of evaporation were carried out under reduced pressure in an BUCHI rotary vacuum
evaporator at bath temperature not exceeding 45 ºC. The residual solvent in the extract and
compounds were removed under high vacuum.
Fig 2.1 : Rotary Evaporator (BUCHI)
21 | P a g e
2.1.4. Freeze-drying
All freeze-drying were performed with a Varian 801 model LY-3-TT and HETOSICC (Denmark) freeze-
dryer.
2.2 Preparation of Extracts
The Bael Pulp & Kurchi Bark were collected and washed with water to remove mud and dust particles.
They were first dried in room temperature and then in the oven at 400 C. The dried parts were grind to
powder by a grinder. The powder was stored for extracts in air tight bottle.
Fig 2.2 : Powdered Kurchi Bark (Holarrhena anti-dysenteria)
2.2.1 Initial extraction by Decoction Method
Initial extraction was done by decoction method. The two raw materials subjected to extract were
taken according to the general formula of USP XII.
2.2.1.1 Extraction of BAEL PULP (Aegle marmelos)
50.10g of powdered extract of Bael pulp was taken in a 2000ml beaker. Then 1250ml of cold water was
poured into the beaker followed by boiling for about 2 hours. After that the solution was allowed to
cool to 400C. After achieving the desired temperature the solution was filtered by suction pump.
22 | P a g e
Fig 2.3 : Freeze Dried extract of Bael Pulp (Aegle marmelos)
2.2.1.2 Extraction of KURCHI BARK (Holarrhena anti-dysenteria)
50.09g of powdered extract of Kurchi bark was taken in a 2000ml beaker. Then 1150ml of cold water
was poured into the beaker followed by boiling for about 2 hours. After that the solution was allowed
to cool to 400C. After achieving the desired temperature the solution was filtered by suction pump.
Fig 2.4 : Freeze Dried extract of KURCHI BARK (Holarrhena anti-dysenteria)
2.2.1.3 Evaporation
The two water extract were concentrated using rotary evaporator (BUCHI) at temperature 480C &
72mbar of pressure.
23 | P a g e
2.2.1.4 Freeze Drying
The concentrated solutions were then subjected to freeze drying (Varian 801 model LY-3-TT and HETOSICC
(Denmark) freeze-dryer). Water was completely removed by evaporation and using a drying pumps
before freeze-drying.
That thing was done in different steps. They were as below:
Pre-freezing : In this step all were frozen up to -370C.
Primary Drying : In that step more that 90% water molecules changed directly from solid state to vapor
through sublimation.
Secondary Drying : The residual water molecules remained adsorbed on the product as moisture. They
were desorbed during secondary drying to attain a moisture level too low to permit any biological
growth or chemical reaction while preserving the activity & integrity of the freeze dried product.
Then the dried powdered freeze dried materials were kept in air tight bottle and used for further
analysis.
2.3 Chromatographic Techniques
Chromatography is the collective term for a set of laboratory techniques for the separation of
mixtures. The mixture is dissolved in a fluid called the "mobile phase", which carries it through a
structure holding another material called the "stationary phase". The various constituents of the
mixture travel at different speeds, causing them to separate. The separation is based on differential
partitioning between the mobile and stationary phases.
For separation of extracted compounds into individual pure ones, various types of chromatographic
techniques were used, such as column chromatography, thin layer chromatography (TLC) , paper
chromatography and vacuum liquid chromatography (VLC).
2.3.1 Thin Layer Chromatography (TLC)
Thin layer chromatography (TLC) is a widely employed laboratory technique. it involves a stationary
phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate.
Compared to paper, it has the advantage of faster runs, better separations, and the choice between
different adsorbents. For even better resolution and to allow for quantification, high-performance TLC
can be used.
24 | P a g e
For this thesis purpose commercially available pre-coated silica gel (Kiesel gel 60 PF254) plates were
used.
2.3.2 Sample application (spotting the plates)
The TLC plates were spotted with a small amount of the crude extract by using a narrow glass capillary.
The capillary was washed with either acetone or ethanol before each sample was applied.
Fig 2.5 : Spotting of TLC plate
2.3.3 Preparation of TLC tank
A small spot of the solution is applied on the activated silica plate with a capillary tube just 1 cm above
the lower edge of the plate. The spot is air dried and a straight line is drawn 2 cm below the upper
edge of the activated plate which marks the upper limit of the solvent flow.
The spotted plate is then placed in the beaker in such a way as to keep the applied spot above the
surface of the solvent system and the lid is placed again. The plate is left for development.
When the solvent front reaches up to the given mark, the plate is taken out and air-dried.
Fig 2.6 : TLC tank
25 | P a g e
2.3.4 Solvent Systems
The solvents of different polarity used for TLC were given below:
Chloroform: Methanol: Water : Acetic Acid (7:2:0.5:0.5)
2.3.5 Visualization/Detection of Compounds
For the location of the separated components, the TLC plates were examined by using the method in
which the plates were sprayed with vanillin-sulfuric acid regent (1.0%) followed by heating in an oven
at 1100C for 10 minutes.
2.3.6 Vanillin-sulphuric acid reagent
Vanillin (1.0 g) was added to the sulfuric acid (100 ml) (kept in ice bath), cooled and used for spraying
the TLC plates.
2.3.7 Determination of Rf (Retention factor) Values
The retention factor, or Rf, is defined as the distance traveled by the compound divided by the distance
traveled by the solvent.
Rf value is characteristic of a compound in a specific solvent system. It helps in the identification of
compounds. Rf value of a compound can be calculated by the following formula:
Rf =
26 | P a g e
Figure 2.7: A Plate for the calculation of R f value
The Rf can provide corroborative evidence as to the identity of a compound. If the identity of a
compound is suspected but not yet proven, an authentic sample of the compound, or standard, is
spotted and run on a TLC plate side by side (or on top of each other) with the compound in question. If
two substances have the same Rf value, they are likely (but not necessarily) the same compound. If
they have different Rf values, they are definitely different compounds. Note that this identity check
must be performed on a single plate, because it is difficult to duplicate all the factors which influence Rf
exactly from experiment to experiment.
2.3.8 Physiochemical Screening
The extracts were analyzed for the presence of alkaloids, terpenoids, reducing sugars, saponins,
tannins, carbonyls, flavonoids,phlobatannis and steroids.
2.3.8.1 Test for Alkaloids
2g of freeze dried extract was warmed for 2 minutes with 20 ml 1% H2SO4 in a 50ml conical flask on a
water bath with intermittent shaking, centrifuged ; pipette off the supernatant into a conical flask.
Then one drop of Mayer’s reagent was added with 0.1ml of supernatant in a semi micro tube. And
observed for cream precipitate.
Compound
Compound
Baseline
Solvent front
Distance from sample front, B
27 | P a g e
2.3.8.1.1 Preparation of Mayer’s reagent
It was prepared by dissolving 1.36 g of mercuric chloride in 20 ml distilled water (A) & 5g of potassium
iodide in 10 ml of distilled water (B). A & B were mixed together and the volume was adjusted to 100ml
with distilled water.
2.3.8.2 Test for Cardiac glycoside
Keller-Killani Test
5g of freeze dried extract was taken in a separate test tube with 2 ml of glacial acetic acid containing a
drop of ferric chloride solution. This was under layered with 1 ml of concentrated sulfuric acid. And
observe for brown ring formation at the interface (Finar, 1983).
2.3.8.3 Test for Terpenoids
5g of freeze dried extract was taken in separate test tubes with 2 ml of chloroform. And
concentrated H2SO4 was added carefully to form a layer. And observed for presence of reddish brown
color interface to show positive results for the presence of terpenoids.
2.3.8.4 Test for Saponins
About 2 g of the powdered sample was boiled in 20 ml of distilled water in a water bath and was
filtered. 10 ml of the filtrate was mixed with 5ml of distilled water and was shaken vigorously for a
stable persistent froth. Frothing was mixed with 3 drops of olive oil and was shaken vigorously, then
was observed for the formation of emulsion.
2.3.8.5 Test for Tannins
About 5 g of the dried sample was boiled in 20ml of distilled water in a test tube and was then filtered.
A few drops of 0.1% FeCl3 was added and was observed for brownish green or a blue black coloration.
2.3.8.6 Test for Flavonoids
About 2 g of the powdered sample was boiled in 20 ml of distilled water in a water bath and was
filtered. 10 ml of the filtrate was taken in a test tube and 5ml of diluted ammonia followed by few
drops of con.H2SO4 were added. A yellow coloration was ovserved.Upon further standing the yellow
coloration was disappeared.
28 | P a g e
2.3.8.7 Test for Steroids
2 ml of acetic anhydride was added with 0.5 gm of extract of each sample followed by addition of 2 ml
of Sulphuric acid and observed for the color change from violet to blue or green in samples indicating
the presence of steroids
2.3.8.8 Test for Phlobatannins
About 2 g of the powdered sample was boiled in 20 ml of distilled water in a water bath and was
filtered. 10 ml of the filtrate was taken in a test tube and was boiled with 1% aqueous HCl and
observed for red precipitation.
2.4 Spectroscopic Techniques
2.4.1 Infra-red (IR) Spectroscopy
Infrared spectroscopy (IR spectroscopy) is the spectroscopy that deals with the infrared region of the
electromagnetic spectrum, that is light with a longer wavelength and lower frequency than light. As
with all spectroscopic techniques, it can be used to identify and study chemicals.
Fig 2.8 : Schematics of a two-beam absorption spectrometer
A Shimadzu IR-470 spectrometer was used to record the infra-red spectrum (KBr pellet)
Fig 2.9 : Shimadzu IR-470 spectrometer
29 | P a g e
2.4.1.1 Sample Preparation
I had got both solid (raw materials) and liquid (commercial) samples.
Solid samples were crushed with an oily mulling agent (Nujol) in agate mortar, with a pestle. A thin film
of the mull was smeared onto salt plates and measured. This mixture was then pressed in a mechanical
press to form a translucent pellet through which the beam of the spectrometer can pass.
Liquid samples were sandwiched between two plates of a KBr salt. The plates are transparent to the
infrared light and do not introduce any lines onto the spectra.
2.7.2 Ultra-violet (UV) Spectroscopy
Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or UV/Vis) refers to
absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region. This
means it uses light in the visible and adjacent (near-UV and near-infrared (NIR)) ranges. The absorption
or reflectance in the visible range directly affects the perceived color of the chemicals involved. In this
region of the electromagnetic spectrum, molecules undergo electronic transitions.
Fig 2.10 : Diagram of a single-beam UV/Vis spectrophotometer.
Ultra-violet (UV) spectra were recorded using the Shimadzu UV-160A spectrometer.
30 | P a g e
Fig 2.11: Shimadzu UV-160A spectrometer
Sample Preparation
A small amount of sample was dissolved in a specified solvent to which it was dissolved and the
solution was taken in quartz cell (1cm x1cm) to record the spectrum. The main bands (λ max) were
recorded as wavelength (nm).
2.5 Ash Content Analysis
The ash content is the percentage of inorganic residue remaining after ignition of sample.
The ash content of the two raw materials (Bael pulp & Kuchi Bark) and three market samples (Ibne
Sina, New Life ,Hamdard) was determined by heating them in a muffle furnace (Barnstead Thermolyne,
Model-48000) at 450OC for three hours.
Fig 2.12 : Muffle Furnace(Barnstead Thermolyne, Model-48000)
31 | P a g e
2.5.1 Flame Photometer
Flame photometry, more properly called flame atomic emission spectrometry, is a fast, simple, and
sensitive analytical method for the determination of trace metal ions in solution. Because of the very
narrow and characteristic emission lines from the gas-phase atoms in the flame plasma, the method is
relatively free of interferences from other elements. Typical precision and accuracy for analysis of
dilute aqueous solutions are about ±1-5% relative.
Fig 2.13 : Flame Photometer (JENWAY,Model = PFP7 Flame Photometer)
2.5.1.1 Determination of Sodium (Na) Content by Flame Photometer
The amount of Na content in ash was determined by following ISO standard. The flame photometer
used to determined this is belongs to JENWAY (Model = PFP7 Flame Photometer).That experiment was
done in a temperature and humidity controlled zone.
1 ppm Na spike solution was used to carry out this operation according to ISO.
2.5.1.2 Determination of Potassium (K) Content by Flame Photometer
The amount of K content in ash was determined by following ISO standard. The flame photometer
used to determined this is belongs to JENWAY (Model = PFP7 Flame Photometer).That experiment was
done in a temperature and humidity controlled zone.
1 ppm Na spike solution was used to carry out this operation according to ISO.
2.5.2 Determination of Heavy Metals Using Atomic Absorption Spectroscopy (AAS)
Using herbs in medical treatment of various illnesses one should be aware that apart from the
pharmacological effect they could turn out to be toxic because of the presence of heavy metals like Pb,
32 | P a g e
Cd, Zn, Ni and other impurities. For these reasons it is essential to measure the level of contaminants in
medicinal raw materials.
2.5.2.1 Atomic Absorption Spectroscopy (AAS)
Atomic absorption spectroscopy (AAS) is a spectro analytical procedure for the qualitative and
quantitative determination of chemical elements employing the absorption of optical radiation (light)
by free atoms in the gaseous state. In analytical chemistry the technique is used for determining the
concentration of a particular element (the analyte) in a sample to be analyzed.
The technique makes use of absorption spectrometry to assess the concentration of an analyte in a
sample. It requires standards with known analyte content to establish the relation between the
measured absorbance and the analyte concentration and relies therefore on Beer-Lambert Law. In
short, the electrons of the atoms in the atomizer can be promoted to higher orbitals (excited state) for
a short period of time (nanoseconds) by absorbing a defined quantity of energy (radiation of a given
wavelength). This amount of energy, i.e., wavelength, is specific to a particular electron transition in a
particular element. In general, each wavelength corresponds to only one element, and the width of an
absorption line is only of the order of a few picometers (pm), which gives the technique its elemental
selectivity. The radiation flux without a sample and with a sample in the atomizer is measured using a
detector, and the ratio between the two values (the absorbance) is converted to analyte concentration
or mass using Beer-Lambert Law.
Fig 2.14 : Atomic Absorption Spectrometer block diagram
33 | P a g e
2.5.2.2 Determination of Lead (Pb), Copper (Cu),Cadmium (Cd) & Chromium ( Cr)
The amounts of those heavy metals in ash were determined by following ISO standard. The AAS used
to determined this was belongs to Varian (Model = AA240FS-Fast Sequential Atomic Absorption
Spectrometer).That machine was calibrated on 14th July,2011.That experiment was done in a
temperature and humidity controlled zone.
Here Deuterium background correction technique had been used. That machine was cleaned by 2%
HNO3.
Fig 2.15 : Atomic Absorption Spectrometer (Varian ,Model = AA240FS-Fast Sequential Atomic
Absorption)
2.5.2.3 Determination of Manganese (Mn), Cobalt (Co), Nickel (Ni) & Zinc (Zn)
The amounts of those heavy metals in ash were determined by following ISO standard. The AAS used
to determined this was belongs to Varian (Model = AA240FS-Fast Sequential Atomic Absorption
Spectrometer).That machine was calibrated on 14th July,2011.That experiment was done in a
temperature and humidity controlled zone.
Here Deuterium background correction technique had been used. That machine was cleaned by 2%
HNO3.
2.5.2.4 Determination of Calcium (Ca)
The AAS used to determined this metal was belongs to Shimadzu (Model =AA6401F).That machine
was calibrated on 14th July,2011.That experiment was done in a temperature and humidity controlled
zone.
Here Deuterium background correction technique had been used. That machine was cleaned by 2%
HNO3.
34 | P a g e
Fig 2.16 : Atomic Absorption Spectrophotometer (Shimadzu Model =AA6401F)
2.5.2.4 Determination of Arsenic (As) & Mercury (Hg)
The amounts of those two heavy metals in ash were determined by following ISO standard. The AAS
used to determined this was belongs to Varian (Model = AA240FS-Fast Sequential Atomic Absorption
Spectrometer).Also there was an extended accessories. That was Vapor Generation Accessories (Model
= VGA-77).That machine was calibrated on 14th July,2011.That experiment was done in a temperature
and humidity controlled zone.
Here Deuterium background correction technique had been used. That machine was cleaned by 2%
HNO3.
Fig 2.17 : AAS with VGA Fig 2.18 : Vapor Generation Accessories (Model VGA-77)
35 | P a g e
Experimental Section
3.1 Collection and Preparation of pulp of Aegle marmelos
The pulp of Aegle marmelos was collected from market located in Nawabpur. The pulps were
washed with water to remove mud and dust particles and then in an oven at 400 C
The dried stems were grinded to powder by a cyclotec grinder (200 meshes) and the powder
was stored in an air tight bottle and this was used throughout the investigations.
3.2 Collection and Preparation of bark of Holarrhena anti-dysenteria
The bark of Holarrhena anti-dysenteria was collected from market located in Nawabpur. The
barks were washed with water to remove mud and dust particles and then in an oven at 400 C
The dried stems were grinded to powder by a cyclotec grinder (200 meshes) and the powder
was stored in an air tight bottle and this was used throughout the investigations.
3.3 Collection of Commercial Samples
Three commercial samples were collected from market belongs to three different manufacturer. They
were from Ibne Sina (Bel Syrup),New Life (Syrup anti-dysenteria) & Hamdard (Syrup Marbelus). All of
their exp. date had been checked.
Fig 3.1 : Commercial samples
36 | P a g e
3.4 Determination of Foreign matter
3.4.1 Procedure
Definite amount of each sample was taken and was spreaded in a thin layer.Then the foreign matters
were sorted out into groups by visual inspection followed by using sieve. After passing through the
sieves the remaining samples were weighted.
3.4.2 Experimental data
1. Bael Pulp (Aegle marmelos)
Raw weight
(g)
Final weigh
(g)
Amount of foreign
matter (g)
Percentage of foreign
matter (%)
227.56 226.94 0.62 0.27
Calculation of foreign matter = 10056.227
62.0
= 0.27 %
2. Kurchi Bark (Holarrhena anti-dysenteria)
Raw weight
(g)
Final weigh
(g)
Amount of foreign
matter (g)
Percentage of
foreign material
342.22g 324.6g 17.62g 5.15
Calculation of foreign matter : 10022.342
62.17
= 5.15 %
37 | P a g e
3.6 Determination of Moisture Content
3.5.1 Procedure
Definite amount of each sample was taken in a Petri dish. It was then subject to heat in an
oven for 6 hours at 1100C. After vigorous heating then it was weighted and again heat was
applied for 3 hours. The sample was subject to heat until constant weight was achieved.
3.5.2 Experimental data
Bael Pulp (Aegle marmelos)
Calculation of moisture content: 10094.226
35.20
= 8.97 %
Kurchi Bark (Holarrhena anti-dysenteria)
Initial weigh 1st weigh 2nd weigh Constant
weigh
Amount of
moisture
content
Percentage
of moisture
content
(%)
324.6 288.03 287.14 287.14 0.89 0.27
Initial
Weigh
(g)
1st weigh(g) 2nd
weigh(g)
3rd
weigh(g)
Constant
weigh(g)
Amount
of
moisture
content(g)
(%)
percentage
of moisture
content
226.94 209.14
(at 10:30
pm)
206.63 (at
1:15 am
206.59
(at 3:30
am)
206.59 20.35 8.97
38 | P a g e
Calculation of moisture content: 1006.324
89.0
= 0.27 %
3.6 Determination of Ash content
3.6.1 Procedure
Accurately weighted amount of air dried samples were placed in a previously ignited and tarred silica
crucible. The materials were spreaded in an even layer and it was ignited gradually to 500–600 °C until
it was white, indicating the absence of carbon. It was Cooled in a desiccators and weighted.
Whenever carbon free ash was not observed in that way then the burned sample was moistened with
about 2 ml of acid. Then placed in a hot plate and ignited to constant weigh. Allowed the residue to
cool in suitable desiccators for 30 minutes, then it was weighted without delay.
Fig 3.2 : Ash of raw materials
3.6.2 Experimental data
Bael Pulp (Aegle marmelos)
Weigh of
crucible +
Sample
Weigh of
crucible
(g)
Weigh of
sample
(g)
Weigh of
crucible +
Sample (After
Weigh of Ash
(g)
(%)
Percentage of
Ash
39 | P a g e
(g) burn) (g)
46.6989 42.0879 4.611 42.2554 0.1675 3.63
Calculation of Ash = 100611.4
1675.0
= 3.63 %
Kurchi Bark (Holarrhena anti-dysenteria)
Weigh of
crucible +
Sample
(g)
Weigh of
crucible
(g)
Weigh of
sample
(g)
Weigh of
crucible +
Sample (After
burn)
(g)
Weigh of Ash
(g)
(%)
Percentage of
Ash
43.6206 38.8760 4.7446 39.1951 0.3191 6.73
Calculation of Ash = 1007446.43191.0
= 6.73 %
Bael Syrup (Ibne Sina)
Weigh of
crucible
(g)
Volume of
Sample
(ml)
Weigh of
crucible +
Sample (After
burn)
(g)
Weigh of Ash
(g)
(%) Percentage
of Ash
30.4162 50.0 30.6158 0.1996 0.3992
40 | P a g e
Syrup Anti-dysenteria (New Life)
Weigh of
crucible
(g)
Volume of
Sample
(ml)
Weigh of
crucible +
Sample (After
burn)
(g)
Weigh of Ash
(g)
(%) Percentage
of Ash
43.5464 50.0 43.7874 0.241 0.482
Syrup Marbelos (Hamdard)
Weigh of
crucible
(g)
Volume of
Sample
(ml)
Weigh of
crucible +
Sample (After
burn)
(g)
Weigh of Ash
(g)
(%) Percentage
of Ash
73.7835 50.0 74.0766 0.2931 0.5862
Fig 3.3 : Ash of commercial samples
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3.7 Determination of Extractable Matter (Decoctions method)
3.7.1 Extraction Procedure of Bael Pulp (Aegle marmelos)
50.10g of powdered extract of Bael pulp was taken in a 2000ml beaker. Then 1250ml of cold water was
poured into the beaker followed by boiling for about 2 hours. After that the solution was allowed to
cool to 400C. After achieving the desired temperature the solution was filtered by suction pump using
Whatman 40 filter paper. After filtration the filtrate was concentrated using rotary evaporator (BUCHI).
In rotary evaporator the water was removed using 72mbar of pressure and 450C.After evaporation the
thick concentrated pulp extract was subject to freeze drying(Varian 801 model LY-3-TT and HETOSICC
(Denmark) freeze-dryer). Water was completely removed by evaporation and using a drying pumps
before freeze-drying.
This was done in different steps. They are as below:
Pre-freezing : In this step all were frozen up to -350C.
Primary Drying : In that step more that 90% water molecules changed directly from solid state to vapor
through sublimation.
Secondary Drying : The residual water molecules remained adsorbed on the product as moisture. They
were desorbed during secondary drying to attain a moisture level too low to permit any biological
growth or chemical reaction while preserving the activity & integrity of the freeze dried product.
Then the dried powdered freeze dried materials were weighted and kept in a fresh bottle.
3.7.2 Experimental data
Weight of empty bottle = 3.3185g
Weight of empty bottle + Sample (After freeze drying)= 26.1245g
Weight of extractable matter = 22.806g
42 | P a g e
Fig 3.4 : Extractable Matter of Bael Pulp (Aegle marmelos)
3.7.3 Extraction Procedure of Kurchi Bark (Holarrhena anti-dysenteria)
50.09g of powdered extract of Kurchi bark was taken in a 2000ml beaker. Then 1150ml of cold water
was poured into the beaker followed by boiling for about 2 hours. After that the solution was allowed
to cool to 400C. After achieving the desired temperature the solution was filtered by suction pump
using Whatman 40 filter paper. After filtration the filtrate was concentrated using rotary evaporator
(BUCHI).
In rotary evaporator the water was removed using 72mbar of pressure and 450C.After evaporation the
thick concentrated bark extract was subject to freeze drying(Varian 801 model LY-3-TT and HETOSICC
(Denmark) freeze-dryer). Water was completely removed by evaporation and using a drying pumps
before freeze-drying.
This was done in different steps. They are as below:
Pre-freezing : In this step all were frozen up to -350C.
Primary Drying : In that step more that 90% water molecules changed directly from solid state to vapor
through sublimation.
Secondary Drying : The residual water molecules remained adsorbed on the product as moisture. They
were desorbed during secondary drying to attain a moisture level too low to permit any biological
growth or chemical reaction while preserving the activity & integrity of the freeze dried product.
Then the dried powdered freeze dried materials were weighted and kept in a fresh bottle.
3.7.4 Experimental data
Weight of empty bottle = 3.2662g
43 | P a g e
Weight of empty bottle + Sample (After freeze drying) = 8.8235g
Weight of extractable matter = 5.5573g
Fig 3.5 : Extractable Matter of Kurchi Bark (Holarrhena anti-dysenteria)
44 | P a g e
Ash Content Analysis 4.3 Determination of Sodium (Na) content by Flame Photometer
4.3.1 Apparatus
Jenway Flame Photometer, Model PFP7 Flame Photometer, with a total-consumption burner using
natural gas and oxygen. Wavelength isolation was by use of a simple interference filter. Light from the
flame was focused onto the end of a fiber optic cable (a "light pipe") which transmits the light onto a
photodiode in the small electronics box by the flame photometer. The electronics there converted the
diode's output into a digital display.
Fig 4.1 : Flame Photometer (JENWAY,Model = PFP7 Flame Photometer)
4.3.2 Solution Preparation
For preparing 1000 ppm Na stock solution 2.542 g of dried pure NaCl was dissolved in 1 liter of
deionised water.
From that stock solution, 1 ppm, 2 ppm & 4 ppm Standard Na solution was prepared.
4.3.3 Experimental Data
ID Amount of
Ash
Dilution Absorption Amount in
ppm
Amount in
mg/kg
%
Bael Pulp 0.1675 5 5.45 3.1774*5 95 0.95
Kurchi
Bark
0.3191 5 4.80 2.7430*5 43 0.43
Ibne Sina 0.1996 200 3.77 2.0924*200 2100 21.0
45 | P a g e
New Life 0.241 200 4.1 2.296*200 1900 19.0
200
(Duplicate)
4.0 2.2340*200 1854 18.54
200+1
ppm Na
Spike
5.83 3.4411-1*200 2026 20.26
Hamdard 0.2931 200 4.14 2.3212*200 1584 15.84
Std. 1 ppm Na = 1.90
Std. 2 ppm Na = 3.61
Std. 4 ppm Na = 6.60
4.3.4 Calibration Curve
Fig 4.2: Calibration curve for Sodium (Na)
0
1
2
3
4
5
6
7
0 1 2 3 4 5
Absorbance
Concentration
Calibration Curve
Calibration Curve
Linear (Calibration Curve)
46 | P a g e
4.4 Determination of Potassium (K) content by Flame Photometer
4.4.1 Apparatus
Jenway Flame Photometer, Model PFP7 Flame Photometer, with a total-consumption burner using
natural gas and oxygen. Wavelength isolation was by use of a simple interference filter. Light from the
flame was focused onto the end of a fiber optic cable (a "light pipe") which transmits the light onto a
photodiode in the small electronics box by the flame photometer. The electronics there converted the
diode's output into a digital display.
4.4.2 Solution Preparation
From 1000 ppm K stock solution 1 ppm, 2 ppm & 4 ppm Standard K solution was prepared.
4.4.3 Experimental Data
ID Amount
of Ash
Dilution Absorbance Amount in
ppm
Amount
in mg/kg
%
Bael Pulp 0.1675 250 5.8 1.8829*250 2810 28.1
250
(Duplicate)
5.5 1.7804*250 2657 26.57
250+1
ppm Na
Spike
8.14 2.7036-1*250 2543 25.43
Kurchi Bark 0.3191 250 5.80 1.8829*250 1480 14.8
Ibne Sina 0.1996 100 8.88 2.9719*100 1489 14.89
New Life 0.241 50 7.10 2.3339*50 484 4.84
Hamdard 0.2931 50 10.35 3.5184*50 600 6.0
Std. 1 ppm Na = 3.13
Std. 2 ppm Na = 6.16
Std. 4 ppm Na = 11.60
4.4.4 Calculation
ଵ.଼଼ଶଽ×ଶହ×ଵଵ×.ଵହ×ଵ
= 2810 mg/Kg
47 | P a g e
4.2.5 Calibration Curve
Fig 4.3: Calibration curve for Potassium (K)
4.3 Determination of Heavy Metals by Atomic Absorption Spectrometry (AAS)
4.3.1 Determination of Palladium (Pd), Copper (Cu),Cadmium (Cd) & Chromium ( Cr)
4.3.1.1 Apparatus
The amounts of those heavy metals in ash were determined by following ISO standard. The AAS used
to determined this was belongs to Varian (Model = AA240FS-Fast Sequential Atomic Absorption
Spectrometer).
Fig 4.4 : Atomic Absorption Spectrometer (Varian ,Model = AA240FS-Fast Sequential Atomic
Absorption)
0
5
10
15
0 2 4 6
Absorbance
Concentration
Calibration Curve
Calibration Curve
Linear (Calibration Curve)
48 | P a g e
Machine Details
Model: AA240FS-Fast Sequential Atomic Absorption Spectrometer
Machine ID: AA240FS
Manufacturer: Varian
Calibration Date: 14 July, 2011
4.3.1.2 Solution Preparation
For the analysis of Pd,Cd,Cr & Cu my reference solutions were 2.0 ppm Pd,0.4 ppm Cd, 2.0 ppm Cr &
2.0 ppm Cu. My stock solutions were all 1000 ppm. From those stock solutions I made the required
ppm solutions by applying the formula V1S1 = V2S2
4.3.1.3 Experimental Data
Samples Labels Cr 357.9nm
ppm
Cu 324.8nm
ppm
Cd 228.8nm
ppm
Pd 217.0
ppm
QC.STD.No.2 0.650 0.505 0.100 0.51
Bael Pulp -0.005 -0.002 -0.001 0.01
Kurchi Bark -0.029 0.002 -0.001 0.02
Ibne Sina 0.033 0.050 0.002 0.17
New Life 0.110 0.344 0.001 0.05
Hamdard 0.056 OVER 0.002 0.10
QC.STD.No.2 0.280 0.471 0.094 0.49
QC.STD.No-2
1. 2.0 ppm Pb
2. 0.4 ppm Cd
3. 2.0ppm Cr
4. 2.0 ppm Cu
49 | P a g e
4.3.2 Determination of Manganese (Mn), Cobalt (Co), Nickel (Ni) & Zinc (Zn)
4.3.2.1 Apparatus
The amounts of those heavy metals in ash were determined by following ISO standard. The AAS used
to determined this was belongs to Varian (Model = AA240FS-Fast Sequential Atomic Absorption
Spectrometer).
Machine Details
Model: AA240FS-Fast Sequential Atomic Absorption Spectrometer
Machine ID: AA240FS
Manufacturer: Varian
Calibration Date: 14 July, 2011
4.3.2.2 Solution Preparation
For the analysis of Mn, Co, Ni & Zn my reference solutions were 0.5 ppm Ni, 0.3 ppm Zn, 0.5 ppm Mn &
0.5 ppm Co. My stock solutions were all 1000 ppm. From those stock solutions I made the required
ppm solutions by applying the formula V1S1 = V2S2
4.3.2.3 Experimental Data
Samples Labels Mn279.5nm
mg/L
Co 240.7nm
mg/L
Ni 232.0nm
mg/L
Zn 213.9nm
mg/L
QC.STD.No.2 0.453 0.503 0.504 0.3032
Bael Pulp -0.024 0.017 0.002 0.0019
Kurchi Bark -0.027 0.013 0.002 0.0028
Ibne Sina 0.489 0.013 0.081 0.2674
New Life 0.231 0.011 0.374 0.8221
Hamdard 1.923 0.956 0.926 0.9007
QC.STD.No.2 0.413 0.499 0.479 0.2887
QC.STD.No-2
1. 0.5ppm Ni
2. 0.3ppm Zn
50 | P a g e
3. 0.5ppm Mn
4. 0.5ppm Co
4.3.3 Determination of Calcium (Ca)
4.3.3.1 Apparatus
The amounts of Calcium in ash were determined by following ISO standard. The AAS used to
determined this was belongs to Shimadzu (Model = AA6401F )
Fig 4.5 : Atomic Absorption Spectrophotometer (Shimadzu Model =AA6401F)
Machine Details
Model: AA6401F
Manufacturer: Shimadzu
Calibration Date: 14 July, 2011
4.3.3.2 Solution Preparation
Stock solution was 1000 ppm. From that using V1S1=V2S2 I made these solutions. First I prepared
500ppm solution. Then from 500 ppm solution I made 0.5,1.0,2.0 ppm solution.
Preparing 10ppm solution
V1S1=V2S2
1000*V1 = 10*500
V1 = 5ml
Take 5ml & up to the mark in 500ml volumetric flask. This is 10ppm solution. From This I prepared
0.5,1.0,2.0 ppm solutions
51 | P a g e
First after running QC samples I had to check whether my absorbance would be within the range or
not. So I had to take 2.0ppm solution and read the absorbance. Then check my samples absorbance
whether they comply with the maximum ppm solutions absorbance or not. I found that 3 of my
samples Abs is out of range. So I had to dilute them.
A-7677-50 times
A 7678- 2.5 times
A 7680-5 times.
Dilution of A 7677 (50 times)
Pipette out 2ml of sample & put it into 100 ml volumetric flask. Up to the mark.
Dilution of A 7678 (2.5 times)
Pipette out 10ml of sample & put it into 25 ml volumetric flask. Up to the mark.
Dilution of A 7680 (5 times)
Pipette out 5ml of sample & put it into 25 ml volumetric flask. Up to the mark.
4.3.3.3 Flame/calibration Standard Measurement
STD no Concentration
ppm
Absorbance
422.7nm
1 0.5000 0.0142
2 1.0000 0.0361
3 2.0000 0.0668
52 | P a g e
4.3.3.4 Calibration Curve
Fig 4.6 : Calibration Curve for Manganese (Mn), Cobalt (Co), Nickel (Ni) & Zinc (Zn)
4.3.3.5 Experimental Data
Sample no Concentration
ppm
Absorbance
422.7nm
Bael Pulp 1.3617 0.0461
Kurchi Bark 1.5964*50 0.0538
Ibne Sina 1.3975*2.5 0.0472
New Life 1.1714 0.0397
Hamdard 2.0987*5 0.0704
00.010.020.030.040.050.060.070.08
0 0.5 1 1.5 2 2.5
Absorbance
Concentration
Calibration Curve
Calibration Curve
Linear (Calibration Curve)
53 | P a g e
4.3.4 Determination of Arsenic (As)
4.3.4.1 Apparatus
The amounts of As in ash were determined by following ISO standard. The AAS used to determined this
was belongs to Varian (Model = AA240FS-Fast Sequential Atomic Absorption Spectrometer).
Fig 4.7 : AAS with VGA
Fig 4.8 : Vapor Generation Accessories (Model VGA-77)
Fig 4.9 : Quartz Absorption cell for Arsenic Determination
54 | P a g e
Machine Details
Model: AA240FS-Fast Sequential Atomic Absorption Spectrometer
Machine ID: AA240FS
Manufacturer: Varian
Calibration Date: 14 July, 2011
4.3.4.2 Solution Preparation
Stock solution was 1000 ppb.From that using V1S1=V2S2 I made these solutions. First I prepared 500ppb
solution. Then from 500 ppb solution I made 2ppb, 5ppb, 10ppb, 15ppb & 20 ppb solution.
Fig 4.10 : Solutions used for determining Arsenic Content
4.3.4.3 Reagents
0.6% NaBH4
0.5% NaOH
6 M HCL
4.3.4.4 Procedure
First I took 25 ml beaker and slight water was added followed by addition of 2.5 ml conc. HCl. After
that addition,2.5 ml 1% KI was added to the solution mixture and kept the mixture overnight. Next
morning 0.6 % NaBH4 & 0.5 % NaOH was added. After that the solutions were subjected to AAS.
55 | P a g e
4.3.4.5 Flame/calibration Standard measurement
STD. Absorbance Concentration
Cal Zero -0.0055 0.00
Std-1 0.0593 2.00
Std-2 0.1452 5.00
Std-3 0.2645 10.00
Std-4 0.3682 15.00
Std-5 0.4575 20.00
4.3.4.6 Calibration Curve
Fig 4.11 : Calibration Curve for Arsenic (As)
4.3.4.7 Experimental Data
Sample Label As 193.7nm
ppb (µg/L) Absorbance
QC.10 ppb As (AA) 9.79 0.2625
Bael Pulp 2.14 0.0630
Kurchi Bark -0.34*5 -0.0101
Ibne Sina 0.27*5 0.0081
00.05
0.10.15
0.20.25
0.30.35
0.40.45
0.5
0 5 10 15 20 25
Absorbance
Concentration
Calibration Curve
Calibration Curve
Linear (Calibration Curve)
56 | P a g e
New Life 1.07*5 0.0318
Hamdard 2.63*5 0.0772
QC.10 ppb As (AA) 9.38 0.2532
QC.Method.Blank -0.46 0.0137
4.3.5 Determination of Mercury (Hg)
4.3.5.1 Apparatus
The amounts of Hg in ash were determined by following ISO standard. The AAS used to determined
this was belongs to Varian (Model = AA240FS-Fast Sequential Atomic Absorption Spectrometer).
Machine Details
Model: AA240FS-Fast Sequential Atomic Absorption Spectrometer
Machine ID: AA240FS
Manufacturer: Varian
Calibration Date: 14 July, 2011
4.3.5.2 Solution Preparation
Stock solution was 1000 ppb.From that using V1S1=V2S2 I made these solutions. First I prepared 500ppb
solution. Then from 500 ppb solution I made 5ppb, 10ppb, 20ppb & 40 ppb solution.
4.3.5.3 Reagents
0.3% NaBH4
0.5% NaOH
5 M HCL
4.3.5.4 Procedure
First I took 25 ml beaker and slight water was added followed by addition of 2.5 ml conc. HCl. After
that addition 2.5 ml 1% KI was added to the solution mixture and kept the mixture overnight. Next
morning 0.3 % NaBH4 & 0.5 % NaOH was added. After that the solutions were subjected to AAS.
4.3.5.5 Flame/calibration Standard measurement
STD. Absorbance Concentration
Cal Zero -0.0002 0.00
57 | P a g e
Std-1 0.0991 5.00
Std-2 0.2381 10.00
Std-3 0.4327 20.00
Std-4 0.8841 40.00
4.3.5.6 Calibration Curve
Fig 4.12 : Calibration Curve for Mercury (Hg)
4.3.5.7 Experimental Data
Sample Label Hg 253.7 nm
ppb (µg/L) Absorbance
QC.10 ppb As (AA) 10.83 0.2431
Bael Pulp 0.07 0.0658
Kurchi Bark -0.19 -0.0101
Ibne Sina 0.41 0.0071
New Life -0.01 0.0018
Hamdard 0.55 0.0872
QC.10 ppb As (AA) 10.53 0.2234
00.10.20.30.40.50.60.70.80.9
1
0 10 20 30 40 50
Absrbance
Concentration
Calibration Curve
Calibration Curve
Linear (Calibration Curve)
58 | P a g e
Extractable Matter Analysis
5.1 Thin layer chromatography of the Freeze Dried Extract & Commercial Samples
For the comparison between the raw materials (Bael pulp & Kurchi Bark) and the commercial samples
(Ibne Sina, New Life & Hamdard) they were undergo thin layer chromatography.
A TLC (solvent used as the different ratio of Chloroform, Methanol, Water & Acetic Acid) study of the
decoction extract & the commercial samples dissolved in Methanol showed the presence of spot under
day-light. TLC study showed the presence of distinctive spots being visible on spraying with vanillin-
sulfuric acid followed by heating for 10 minutes.
Of these spots one was violet and one was brown and for commercial samples two spots had been
observed for each one.
Fig 5.1 : Thin layer chromatography of the Freeze Dried Extract & Commercial Samples
5.1.1 Development and determination of the Solvent System
Sample applied : Raw materials & commercial samples
Solvent system : Chloroform: Methanol: Water: Acetic Acid (7 : 2 : 0.5 : 0.5)
The samples were spotted with the help of capillary tube on precoated Aluminium Sheets of Silica Gel
60 F254 (Merck).After trying with various solvent systems with variable volume ratios, the suitable
solvent system as stated above is selected in its proportionate ratio and developed in the chamber of
TLC to the maximum height of the plate so that it can separate the components on the polar phase of
59 | P a g e
silica gel and that of mobile phase of solvent system. Twp raw materials & three commercial
formulation were spotted separately and developed the TLC plate as shown in Figure 5.1.
5.1.2 Procedure
For preparing the solvent system in one measuring cylinder 3.5 ml Chloroform, 1 ml methanol, 0.25 ml
water & 0.25 ml Acetic acid was taken and mixed thoroughly and after that the whole solution was
poured into a beaker.
After spotting the TLC plate was dipped into the beaker in such a way that the spots were way above
the solvent solution. Then the system was covered with watch glass and remained stand still until the
solvents reached up to the above marking of the TLC plate.
Finally the solvent soaked TLC plates were taken outside of the beaker and dried in air for 10 minutes.
After 10 minutes when the plate was completely dried then it was sprayed by developing agent and
kept in oven for 10 minutes. After that I got the following TLC plate.
Fig 5.2: TLC chromatogram of raw & commercial samples
60 | P a g e
5.2 Spectroscopic analysis of the Freeze Dried Extract & Commercial Samples
5.2.1 Infrared Spectroscopy
I had got both solid (decoction extracts) and liquid (commercial) samples.
Solid samples were crushed with an oily mulling agent (Nujol) in agate mortar, with a pestle. A thin film of
the mull was smeared onto salt plates and measured. This mixture was then pressed in a mechanical
press to form a translucent pellet through which the beam of the spectrometer can pass.
Liquid samples were sandwiched between two plates of a KBr salt. The plates are transparent to the
infrared light and do not introduce any lines onto the spectra.
Fig 5.3 : Shimadzu IR-470 spectrometer
61 | P a g e
5.2.1.1 IR Spectra of Bael Pulp (Aegle marmelos)
Fig 5.4 : IR Spectra of Bael Pulp (Aegle marmelos)
5.2.1.1.1 Spectroscopic characteristics
IR spectra of (Fig 5.4) : max cm-1(in KBr pellet)
3510 Carboxylic acid O-H (low concentration)
3410 N-H
3200 Aromatic C-H
2910 Aliphatic or sp3 C-H stretching
1710 C=O
1620, 1566 C=C & aromatic ring system
1450 -CH2- bending in aliphatic compound
1090 C-O stretching
62 | P a g e
5.2.1.2 IR Spectra of Kurchi Bark (Holarrhena anti-dysenteria)
Fig 5.5 : IR Spectra of Kurchi Bark (Holarrhena anti-dysenteria)
5.2.1.2.1 Spectroscopic characteristics
IR spectra of (Fig 5.5) : max cm-1(in KBr pellet)
3410 N-H
3300 Aromatic C-H
2910 Aliphatic or sp3 C-H stretching
1705 C=O
1620, 1590 C=C & aromatic ring system
1450 -CH2- bending in aliphatic compound
1030 C-O stretching
63 | P a g e
5.2.1.3 IR Spectra of Bel Syrup (Ibne Sina)
Fig 5.6 : IR Spectra of Bel Syrup (Ibne Sina)
5.2.1.3.1 Spectroscopic characteristics
IR spectra of (Fig 5.6) : max cm-1(in KBr pellet)
3490 N-H
3320 Aromatic C-H
1720 C=O
1620, 1610 C=C & aromatic ring system
1490 -CH2- bending in aliphatic compound
1050 C-O stretching
64 | P a g e
5.2.1.4 IR Spectra of Syrup anti-dysenteria (New Life)
Fig 5.7 : IR Spectra of Syrup anti-dysenteria (New Life)
5.2.1.4.1 Spectroscopic characteristics
IR spectra of (Fig 5.7) : max cm-1(in KBr pellet)
3560 Carboxylic acid O-H (low concentration)
3490 N-H
3410 Aromatic C-H
1690 C=O
1610, 1505 C=C & aromatic ring system
1490 -CH2- bending in aliphatic compound
1050 C-O stretching
65 | P a g e
5.2.1.5 IR Spectra of Syrup Marbelus (Hamdard)
Fig 5.8 : IR Spectra of Syrup Marbelus (Hamdard)
5.2.1.3.1 Spectroscopic characteristics
IR spectra of (Fig 5.8) : max cm-1(in KBr pellet)
3510 Carboxylic acid O-H (low concentration)
3410 N-H
3210 Aromatic C-H
1690 C=O
1640, 1600 C=C & aromatic ring system
1070 C-O stretching
66 | P a g e
5.2.2 UV Spectroscopy
Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or UV/Vis) refers to
absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region. This
means it uses light in the visible and adjacent (near-UV and near-infrared (NIR)) ranges. The absorption
or reflectance in the visible range directly affects the perceived color of the chemicals involved. In this
region of the electromagnetic spectrum, molecules undergo electronic transitions.
Ultra-violet (UV) spectra were recorded using the Shimadzu UV-160A spectrometer.
A small amount of sample was dissolved in a specified solvent to which it was dissolved and the
solution was taken in quartz cell (1cm x1cm) to record the spectrum. The main bands (λ max) were
recorded as wavelength (nm).
Fig 5.9 : Shimadzu UV-160A spectrometer
67 | P a g e
5.2.2.1 UV Spectra of Bael Pulp (Aegle marmelos)
Fig 5.10 : UV Spectra of Bael Pulp (Aegle marmelos)
68 | P a g e
5.2.2.2 UV Spectra of Kurchi Bark (Holarrhena anti-dysenteria)
Fig 5.11 : UV Spectra of Kurchi Bark (Holarrhena anti-dysenteria)
69 | P a g e
5.2.2.3 UV Spectra of Bel Syrup (Ibne Sina)
Fig 5.12 : UV Spectra of Bel Syrup (Ibne Sina)
70 | P a g e
5.2.2.4 UV Spectra of Syrup anti-dysenteria (New Life)
Fig 5.13 : UV Spectra of Syrup anti-dysenteria (New Life)
71 | P a g e
5.2.2.5 UV Spectra of Syrup Marbelus (Hamdard)
Fig 5.14 : UV Spectra of Syrup Marbelus (Hamdard)
72 | P a g e
5.3 Phytochemical Screening of Raw Materials & Commercial Samples
5.3.1 Phytochemical Screening of Bael Pulp (Aegle marmelos)
The powdered plant material (50.10 gm) was extracted with water using decoction method. The
extract was filtered with a suction pump and the filtrate was concentrated in vacuum evaporator
(BUCHI) followed by freeze dried by Varian 801 model LY-3-TT and HETOSICC (Denmark) freeze-dryer.
Dried extract was used for further studies.
5.3.1.1 Test for Alkaloids
2g of freeze dried extract was warmed for 2 minutes with 20 ml 1% H2SO4 in a 50ml conical flask on a
water bath with intermittent shaking, centrifuged ; pipette off the supernatant into a conical flask.
Then one drop of Mayer’s reagent was added with 0.1ml of supernatant in a semi micro tube. And
observed for cream precipitate.
5.3.1.1.1 Preparation of Mayer’s reagent
It was prepared by dissolving 1.36 g of mercuric chloride in 20 ml distilled water (A) & 5g of potassium
iodide in 10 ml of distilled water (B). A & B were mixed together and the volume was adjusted to 100ml
with distilled water.
5.3.1.2 Test for Cardiac glycoside
Keller-Killani Test
5g of freeze dried extract was taken in a separate test tube with 2 ml of glacial acetic acid containing a
drop of ferric chloride solution. This was under layered with 1 ml of concentrated sulfuric acid. And
observe for brown ring formation at the interface (Finar, 1983).
5.3.1.3 Test for Terpenoids
5g of freeze dried extract was taken in separate test tubes with 2 ml of chloroform. And
concentrated H2SO4 was added carefully to form a layer. And observed for presence of reddish brown
color interface to show positive results for the presence of terpenoids.
5.3.1.4 Test for Saponins
About 2 g of the freeze dried extract was boiled in 20 ml of distilled water in a water bath and was
filtered. 10 ml of the filtrate was mixed with 5ml of distilled water and was shaken vigorously for a
stable persistent froth. Frothing was mixed with 3 drops of olive oil and was shaken vigorously, then
was observed for the formation of emulsion.
73 | P a g e
5.3.1.5 Test for Tannins
About 5 g of freeze dried extract was boiled in 20ml of distilled water in a test tube and was then
filtered. A few drops of 0.1% FeCl3 was added and was observed for brownish green or a blue black
coloration.
5.3.1.6 Test for Flavonoids
About 2 g of the freeze dried extract was boiled in 20 ml of distilled water in a water bath and was
filtered. 10 ml of the filtrate was taken in a test tube and 5ml of diluted ammonia followed by few
drops of con.H2SO4 were added. A yellow coloration was ovserved.Upon further standing the yellow
coloration was disappeared.
5.3.1.7 Test for Steroids
2 ml of acetic anhydride was added with 0.5 gm of freeze dried extract followed by addition of 2 ml
of Sulphuric acid and observed for the color change from violet to blue or green in samples indicating
the presence of steroids
5.3.1.8 Test for Phlobatannins
About 2 g of the freeze dried extract was boiled in 20 ml of distilled water in a water bath and was
filtered. 10 ml of the filtrate was taken in a test tube and was boiled with 1% aqueous HCl and
observed for red precipitation.
5.3.2 Qualitative Phytochemical Screening of Bael Pulp (Aegle marmelos)
Test No. Test Result
1 Alkaloid +
2 Cardiac glycoside +
3 Terpenoids +
4 Saponins +
5 Tannins -
6 Flavonoids +
7 Steroids +
8 Phlobatannins -
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5.3.2 Phytochemical Screening of Kurchi Bark (Holarrhena anti-dysenteria)
The powdered plant material (50.09 g) was extracted with water using decoction method. The extract
was filtered with a suction pump and the filtrate was concentrated in vacuum evaporator (BUCHI)
followed by freeze dried by Varian 801 model LY-3-TT and HETOSICC (Denmark) freeze-dryer. Dried
extract was used for further studies.
5.3.2.1 Test for Alkaloids
2g of freeze dried extract was warmed for 2 minutes with 20 ml 1% H2SO4 in a 50ml conical flask on a
water bath with intermittent shaking, centrifuged ; pipette off the supernatant into a conical flask.
Then one drop of Mayer’s reagent was added with 0.1ml of supernatant in a semi micro tube. And
observed for cream precipitate.
5.3.2.1.1 Preparation of Mayer’s reagent
It was prepared by dissolving 1.36 g of mercuric chloride in 20 ml distilled water (A) & 5g of potassium
iodide in 10 ml of distilled water (B). A & B were mixed together and the volume was adjusted to 100ml
with distilled water.
5.3.2.2 Test for Cardiac glycoside
Keller-Killani Test
5g of freeze dried extract was taken in a separate test tube with 2 ml of glacial acetic acid containing a
drop of ferric chloride solution. This was under layered with 1 ml of concentrated sulfuric acid. And
observe for brown ring formation at the interface (Finar, 1983).
5.3.2.3 Test for Terpenoids
5g of freeze dried extract was taken in separate test tubes with 2 ml of chloroform. And
concentrated H2SO4 was added carefully to form a layer. And observed for presence of reddish brown
color interface to show positive results for the presence of terpenoids.
5.3.2.4 Test for Saponins
About 2 g of the freeze dried extract was boiled in 20 ml of distilled water in a water bath and was
filtered. 10 ml of the filtrate was mixed with 5ml of distilled water and was shaken vigorously for a
stable persistent froth. Frothing was mixed with 3 drops of olive oil and was shaken vigorously, then
was observed for the formation of emulsion.
5.3.2.5 Test for Tannins
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About 5 g of freeze dried extract was boiled in 20ml of distilled water in a test tube and was then
filtered. A few drops of 0.1% FeCl3 was added and was observed for brownish green or a blue black
coloration.
5.3.2.6 Test for Flavonoids
About 2 g of the freeze dried extract was boiled in 20 ml of distilled water in a water bath and was
filtered. 10 ml of the filtrate was taken in a test tube and 5ml of diluted ammonia followed by few
drops of con.H2SO4 were added. A yellow coloration was ovserved.Upon further standing the yellow
coloration was disappeared.
5.3.2.7 Test for Steroids
2 ml of acetic anhydride was added with 0.5 gm of freeze dried extract followed by addition of 2 ml
of Sulphuric acid and observed for the color change from violet to blue or green in samples indicating
the presence of steroids
5.3.2.8 Test for Phlobatannins
About 2 g of the freeze dried extract was boiled in 20 ml of distilled water in a water bath and was
filtered. 10 ml of the filtrate was taken in a test tube and was boiled with 1% aqueous HCl and
observed for red precipitation.
5.3.2 Qualitative Phytochemical Screening of Kurchi Bark (Holarrhena anti-dysenteria)
Test No. Test Result
1 Alkaloid +
2 Cardiac glycoside -
3 Terpenoids -
4 Saponins +
5 Tannins -
6 Flavonoids +
7 Steroids +
8 Phlobatannins -
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5.3.3 Phytochemical Screening of Commercial Samples
5.3.3.1 Test for Alkaloids
2ml of sample was warmed for 2 minutes with 20 ml 1% H2SO4 in a 50ml conical flask on a water bath
with intermittent shaking, centrifuged ; pipette off the supernatant into a conical flask.
Then one drop of Mayer’s reagent was added with 0.1ml of supernatant in a semi micro tube. And
observed for cream precipitate.
5.3.3.1.1 Preparation of Mayer’s reagent
It was prepared by dissolving 1.36 g of mercuric chloride in 20 ml distilled water (A) & 5g of potassium
iodide in 10 ml of distilled water (B). A & B were mixed together and the volume was adjusted to 100ml
with distilled water.
5.3.3.2 Test for Cardiac glycoside
Keller-Killani Test
5ml of sample extract was taken in a separate test tube with 2 ml of glacial acetic acid containing a
drop of ferric chloride solution. This was under layered with 1 ml of concentrated sulfuric acid. And
observe for brown ring formation at the interface (Finar, 1983).
5.3.3.3 Test for Terpenoids
5ml of sample was taken in separate test tubes with 2 ml of chloroform. And concentrated H2SO4 was
added carefully to form a layer. And observed for presence of reddish brown color interface to show
positive results for the presence of terpenoids.
5.3.3.4 Test for Saponins
About 2ml of sample extract was boiled in 20 ml of distilled water in a water bath and was filtered. 10
ml of the filtrate was mixed with 5ml of distilled water and was shaken vigorously for a stable
persistent froth. Frothing was mixed with 3 drops of olive oil and was shaken vigorously, then was
observed for the formation of emulsion.
5.3.3.5 Test for Tannins
About 5 ml of sample extract was boiled in 20ml of distilled water in a test tube and was then filtered.
A few drops of 0.1% FeCl3 was added and was observed for brownish green or a blue black coloration.
5.3.3.6 Test for Flavonoids
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About 2ml of sample was boiled in 20 ml of distilled water in a water bath and was filtered. 10 ml of
the filtrate was taken in a test tube and 5ml of diluted ammonia followed by few drops of con.H2SO4
were added. A yellow coloration was ovserved.Upon further standing the yellow coloration was
disappeared.
5.3.3.7 Test for Steroids
2 ml of acetic anhydride was added with 0.5 ml of sample followed by addition of 2 ml of Sulphuric acid
and observed for the color change from violet to blue or green in samples indicating the presence
of steroids
5.3.3.8 Test for Phlobatannins
About 2ml of sample was boiled in 20 ml of distilled water in a water bath and was
filtered. 10 ml of the filtrate was taken in a test tube and was boiled with 1% aqueous HCl and
observed for red precipitation.
5.3.3 Qualitative Phytochemical Screening of Commercial Samples
Test
no.
Test Bel Syrup (Ibne
Sina)
Syrup anti-
dysenteria (New
Life)
Syrup Marbelus
(Hamdard)
1 Alkaloid + + +
2 Cardiac glycoside - - -
3 Terpenoids + + +
4 Saponins + + +
5 Tannins - - -
6 Flavonoids + + +
7 Steroids + + +
8 Phlobatannins - - -
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Results and Discussion
6.1 Heavy Metal Analysis
The amount of Sodium & Potassium content in both the raw materials (Bael pulp & Kurchi Bark)
& commercial samples (Ibne Sina, New Life & Hamdard) is much greater than any other
metals(Table 6.1). It has a good significance because according to the manufacturer these raw
materials got anti-diarrheal & anti-dysenteric activities. So the amount of Sodium & Potassium
content should be more so that during diarrhea & dysentery these elements can support the
body system by supplying Sodium & Potassium. During diarrhea & Dysentery, the patient is
continuously looses Sodium & Potassium from his body. So in order to balance the amount of
Sodium & Potassium he \she has to take Sodium & Potassium rich items. In this way I think my
raw materials & commercials samples agrees with their claimed activity.
The sodium content found in greater amount in the commercial samples than raw materials
(Table 6.1).I think this was due to the presence of Sodium Benzoate .The chemical formula of
this material is NaC6H5CO2 (Source : Wikipedia).This chemical is worldwide used as preservative
.So it can be assume that all the commercial samples contain this chemical as preservative.
The manufacturers are claiming that their product has got anti-fungal activities. Here from
heavy metal analysis it was found that the amount of copper (Cu) found in market compound is
more than the raw materials (Table 6.1). This chemical element copper has got anti-fungal
activity (Kuhn, P. J. http://www.copper.org/environment/doorknob.html, 1983).
Table 6.1 : Quantitative determination of heavy metals in Raw materials & Commercial Samples
No. Element Name Bael Pulp
(ppm)
Kurchi
Bark
(ppm)
Ibne Sina
(ppm)
New Life
(ppm)
Hamdard
(ppm)
1 Sodium (Na) 15.887 13.715 418.48 459.2 464.24
2 Potassium(K) 445.1 470.725 297.19 116.695 175.92
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3 Mercury (Hg) 0.00007 - 0.00019 0.00041 - 0.00001 0.00055
4 Arsenic (As) 0.00214 - 0.0017 0.00135 0.00535 0.01315
5 Chromium (Cr) -0.005 -0.029 0.033 0.110 0.056
6 Cupper (Cu) -0.002 0.002 0.050 0.344 OVER
7 Cadmium (Cd) -0.001 -0.001 0.002 0.001 0.002
8 Lead (Pb) 0.01 0.02 0.17 0.05 0.10
9 Manganese (Mn) -0.024 -0.027 0.489 0.231 1.923
10 Cobalt (Co) 0.017 0.013 0.013 0.011 0.956
11 Nickel (Ni) 0.002 0.002 0.081 0.374 0.926
12 Zinc (Zn) 0.0019 0.0028 0.2674 0.8221 0.9007
13 Calcium (Ca) 1.3617 79.82 3.49375 1.1714 10.4935
The amount of carcinogenic metals is found to be in safe range. There is a permissible limit of
Arsenic (As). Mercury (Hg), Cadmium (Cd) & Lead (Pb) (Table 6.2). From my heavy metal
detection by AAS it is found that the amount of these metals agreed with the permissible limit
(Table 6.2). So it can be assume that the commercial samples are safe to be taken as medicine.
Table 6.2 : Permissible limit of heavy metals as per WHO
Element
Name
Bael
Pulp
(ppm)
Kurchi
Bark
(ppm)
Ibne
Sina
(ppm)
New
Life
(ppm)
Hamdard
(ppm) Permissible limit as per WHO
Mercury
(Hg) 0.00007 -0.00019 0.00041 -0.00001 0.00055 Not more than 1.0 ppm
Arsenic (As) 0.00214 - 0.0017 0.00135 0.00535 0.01315 Not more than 3.0 ppm
Cadmium
(Cd) -0.001 -0.001 0.002 0.001 0.002 Not more than 0.3 ppm
Lead (Pb) 0.01 0.02 0.17 0.05 0.10 Not more than 10.0 ppm
80 | P a g e
6.2 Extractable Metal Analysis
6.2.1 Thin layer Chromatography Analysis
From the thin layer chromatography analysis we found that the raw materials and two marketed
samples give identical chromatogram but one commercial sample belongs to new life did not give
identical chromatogram like others.
Fig 6.1: TLC Plate
From bael pulp (Aegle marmelos) I got a brown colored spot (Fig 6.1) which has got Rf Value 0.9. The
other raw material Kurchi bark (Holarrhena anti-dysenteria) gave a violet colored spot (Fig 6.1) which
has got Rf value 0.95.
The commercial sample from Ibne Sina (Bel Syrup) gave two identical spots (Fig 6.1)like raw materials.
One spot was brown colored spot which has got Rf value 0.96 & another spot was violet colored which
has got Rf value 0.71.
The commercial sample from Hamdard (Syrup Marbelus) gave two identical spots (Fig 6.1) like raw
materials. One spot was brown colored spot which has got Rf value 0.97 & another spot was violet
colored which has got Rf value 0.81.
Any kind of significant spot was observed from the commercial sample that belongs to New life (Syrup
anti-dysenteria). It may be experimental error.
So from the above discussion I can say that two commercial samples has got the raw materials in their
marketed formulation because each of this kind gave two spots (Fig 6.1) which are identical with the
raw materials (Aegle marmelos & Holarrhena anti-dysenteria). Their Rf value is also close enough.
81 | P a g e
6.2.2 IR Spectroscopic Analysis
6.2.2.1 IR & UV spectroscopic analysis of bael Pulp (Aegle marmelos)
The IR spectrum (Fig 5.4) of the bael Pulp (Aegle marmelos) showed peak at 3510, 3410, 3200,
2910,1710,(1620,1566),1450 & 1090 cm-1 which indicate the presence of carboxylic acid O-H (low
concentration), N-H, Aromatic C-H, Aliphatic or sp3 C-H stretching, >C=0, C=C & aromatic ring system, -
CH2- bending in aliphatic compound,>C-O stretching respectively.
The UV spectrum showed bands at λmax at 286 nm (Fig : 5.10)
6.2.2.2 IR & UV spectroscopic analysis of Kurchi Bark (Holarrhena anti-dysenteria)
The IR spectrum (Fig 5.5) of the Kurchi Bark (Holarrhena anti-dysenteria) showed peak at 3410, 3300,
2910,1705,(1620,1596),1450 & 1030 cm-1 which indicate the presence of N-H, Aromatic C-H, Aliphatic
or sp3 C-H stretching, >C=0, C=C & aromatic ring system, -CH2- bending in aliphatic compound,>C-O
stretching respectively.
The UV spectrum showed bands at λmax at 285 nm (Fig : 5.11)
6.2.2.3 IR & UV spectroscopic analysis of Bel Syrup (Ibne Sina)
The IR spectrum (Fig 5.6) of the Bel Syrup (Ibne Sina) showed peak at 3490, 3320,
1705,(1620,1610),1490 & 1050 cm-1 which indicate the presence of N-H, Aromatic C-H, >C=0, C=C &
aromatic ring system, -CH2- bending in aliphatic compound,>C-O stretching respectively.
The UV spectrum showed bands at λmax at 288 nm (Fig : 5.12)
6.2.2.4 IR & UV spectroscopic analysis of Syrup anti-dysenteria (New life)
The IR spectrum (Fig 5.7) of the Syrup anti-dysenteria (New life) showed peak at 3560, 3490, 3410,
1690,(1610,1505),1490 & 1050 cm-1 which indicate the presence of carboxylic acid O-H (low
concentration), N-H, Aromatic C-H, >C=0, C=C & aromatic ring system, -CH2- bending in aliphatic
compound,>C-O stretching respectively.
The UV spectrum showed bands at λmax at 305 nm (Fig : 5.13)
6.2.2.5 IR & UV spectroscopic analysis of Syrup Marbelus (Hamdard)
The IR spectrum (Fig 5.8) of the Syrup Marbelus (Hamdard) showed peak at 3510, 3410, 3210,
1690,(1640,1600) & 1070 cm-1 which indicate the presence of carboxylic acid O-H (low concentration),
N-H, Aromatic C-H, >C=0, C=C & aromatic ring system,>C-O stretching respectively.
The UV spectrum showed bands at λmax at 293 nm (Fig : 5.14)
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Table 6.3: Comparison table
Name IR Value
(cm-1)
λmax Value
(nm)
Bael Pulp 3510, 3410, 3200, 2910, 1710,
(1620,1566),1450 & 1090
286
Kurchi Bark 3410,3300, 2910, 1705,
(1620,1596),1450 & 1030
285
Ibne Sina 3490, 3320, 1705,
(1620,1610),1490 & 1050
288
New life 3560, 3490, 3410, 1690,
(1610,1505),1490 & 1050
305
Hamdard 3510, 3410, 3210,
1690,(1640,1600) & 1070
293
From the table 6.3 we see that the IR value & the UV absorption value of the raw materials and the
commercial samples are very close to each other. So it can be said that the commercial samples are
containing the raw materials which was claimed by the manufacturers.
Though the materials are present in the commercial samples formulations so the herbal drugs have got
the efficacy of curing dysentery, diarrhea, fungal infections which was claimed by the manufacturers.
The pharmacopeial activities of the drug should be under investigation. In further studies in future it
will be investigated.
6.3 Conclusion
The drug under study was subjected to physicochemical analysis, which is helpful in establishing the
standard along with the other parameters such as heavy metal analysis was done and the presence of
carcinogenic metals were found within the permissible limits of WHO guidelines. Modern analytical
techniques were employed in respect to standardization and to separate the compounds which can be
isolated for further studies. Consequently, the drug was used to determine and ascertain its quality
standard. The study is likely to help in the quality assurance of drug used in the Unani System of
Medicine and in development of standard parameters.
83 | P a g e
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ACRONYMS
ACE angiotension converting enzyme
AGRICOLA The National Agricultural Library Catalogue of the USDA
AIDS acquired immunodeficiency syndrome
API Ayurvedic Pharmacopoeia of India
BCE before Christian era
BHP British Herbal Compendium
CBD Convention on Biological Diversity
CPMP Committee for Proprietary Medicinal Products
EC European Community
EMEA European Medicines Agency
FDA U.S. Federal Drug Administration
GC gas chromatography
HPLC high-performance liquid chromatography
IR Infra red
JSHM Japanese Standards for Herbal Medicines
NMR nuclear magnetic resonance spectroscopy
PC paper chromatography
QC quality control
QA quality assurance
87 | P a g e
TLC thin layer chromatography
UK United Kingdom
USEPA U.S. Environmental Protection Agency
USP U.S. Pharmacopoeia
UV ultraviolet
WTO World Trade Organization