Full Thesis Paper _Office copy_

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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


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


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)



Spectrometer).That machine was calibrated on 14th July,2011.That experiment was done in a

temperature and humidity controlled zone.


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.


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.


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.


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


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

41 | P a g e

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



This was done in different steps. They are as below:







Then the dried powdered freeze dried materials were weighted and kept in a fresh bottle.


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



This was done in different steps. They are as below:







Then the dried powdered freeze dried materials were weighted and kept in a fresh bottle.


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



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. 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



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


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



Spectrometer).

Machine Details


Machine ID: AA240FS




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


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



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)


Dilution of A 7680 (5 times)


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)


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


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


Machine ID: AA240FS




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


Fig 4.11 : Calibration Curve for Arsenic (As)


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


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


Machine ID: AA240FS




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


Fig 4.12 : Calibration Curve for Mercury (Hg)


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


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)



3410 N-H

3300 Aromatic C-H

2910 Aliphatic or sp3 C-H stretching

1705 C=O



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)



3490 N-H

3320 Aromatic C-H

1720 C=O



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)




3490 N-H

3410 Aromatic C-H

1690 C=O



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)




3410 N-H

3210 Aromatic C-H

1690 C=O


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.











Keller-Killani Test









About 2 g of the freeze dried extract was boiled in 20 ml of distilled water in a water bath and was




73 | P a g e


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.







2 ml of acetic anhydride was added with 0.5 gm of freeze dried extract followed by addition of 2 ml







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 -

74 | P a g e

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.











Keller-Killani Test














75 | P a g e

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.







2 ml of acetic anhydride was added with 0.5 gm of freeze dried extract followed by addition of 2 ml







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 -

76 | P a g e

5.3.3 Phytochemical Screening of Commercial Samples


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.








Keller-Killani Test

5ml of sample extract was taken in a separate test tube with 2 ml of glacial acetic acid containing a




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.


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.


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.


77 | P a g e

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.


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


About 2ml of sample was boiled in 20 ml of distilled water in a water bath and was



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 - - -

78 | P a g e

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

79 | P a g e

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

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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.

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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.


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.


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.


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.


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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.

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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

Documents

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