SEMINAR ON

PHYTOCHEMICAL SCREENING AND ESTIMATION OF METALS IN

NATURALLY OCCURRING FRUIT EXTRACTS BY ICP-OES
BY

SHEEMA JABEEN 13411R0083

UNDER THE GUIDANCE OF

MISS. ASRA BEGUMM.PHARM

DEPARTMENT OF PHARMACOLOGY

SHADAN WOMEN’S COLLEGE OF PHARMACY, KHAIRTABAD, HYDERABAD.

 INTRODUCTION 2

• A metal is a word which is derived from Greek called as NOBLE

METALS: Ir,
métallon, "mine, quarry, metal"1,2 . Rh, Pd
• Metal is a material (an element, compound, or alloy)
that is typically hard, opaque, shiny, and has good FERROUS
electrical and thermal conductivity. METALS

HEAVY
METALS:
As, Pb, Ni,
Mn, Cd.

PRECIOUS
METALS:
Pt, Au, Ag.
 TOXICITY 3
 Toxic effects are undesirable disturbances of physiological function caused by poison. A toxic effect can be defined as any
noxious effect on the body – reversible or irreversible, any chemically induced tumor, any teratogenic or mutagenic effect or
death – as a result of contact with a substance via respiratory tract, skin, eye, mouth, or any other route3.
 Possibly required trace elements: Fluorine, Arsenic, Rubidium, Tin, Niobium, Strontium, Gold, Silver and Nickel.
 Toxic metals : Beryllium, Mercury, Lead, Cadmium, Aluminum, Antimony, Bismuth, Barium, Uranium and others.
 Minerals needed in lesser quantities are usually toxic in greater amounts: Copper, Iron, Manganese, Selenium and Vanadium4.
 Even Calcium and Sodium are quite toxic in excess.
 Today mankind is exposed to the highest levels in recorded history of Lead, Mercury, Arsenic, Aluminum, Copper, Nickel, Tin,
Antimony, Bromine, Bismuth and Vanadium.
 Toxic metals sometimes imitate the action of an essential element in the body, interfering with the metabolic process to cause
illness5.
 The toxicity of any metals depends on its ligands.
S.NO ELEMENTS AND BENEFICIAL EFFECTS TOXIC EFFECTS 4
THEIR INTAKE
LEVELS
1. Arsenic Nerve damage, Hyperkeratosis, Pigment change,
Circulatory problems, Cancer risk6

2. Calcium Bone health, treats Osteoporosis, CVS Hypercalcaemia, Renal insufficiency, Kidney
(1000mg-3000mg) disorders, B.P regulation, Cancers of colon stones, Vascular and soft tissue calcification,
rectum, wt. management. Hyperparathyroidism.

3. Cadmium Required for metabolism (in less quantity) Osteomalacia, Osteoporosis, Hypophosphatemia,
(1*10-3 mg/kg/bw) Hyperuricemia7,8, Arthritis, Gout, Muscle
weakness and Coma.

4. Copper Proper growth, prevent premature aging, Hematemesis, Hypotension, Damage to kidneys
(1mg-10mg) regulates Heart rhythms, quick wound healing, and liver, Coma, Jaundice, Gastrointestinal
↓ CH. distress

5. Cobalt Component of Vit B12, formation of Vomiting, Nausea, Cardiomyopathy9, Deafness,

(5µg-8µg) haemoglobin, DNA synthesis, Gp production. Nerve problems, Thyroid problems.
6. Chromium ↑ Insulin production, Damage kidney liver and blood cells, 5
(200µg-5000µg) metabolism and storage of Carbohydrate, Fat Carcinogenic, Weak Immune system10.
and Protein.

7. Iron (Hb) Biological Oxidation, metabolism of ↓Bone marrow, Fatigue, Anemia, Dizziness,
(40mg-45mg) Proteins, Proper growth and development, Asthma, GI disorders, Weak Immune
Strengthen Immune system, Treats system, Ovarian cysts11, Dysmenorrhea
Insomnia, O2 carrier.

8. Manganese Antioxidant, Development, Metabolism, Dopaminergic neuronal death12, inability to

(NMT 10mg) Diabetes, Epilepsy13. walk backwards without falling, oxidative
stress, Mitochondrial dysfunction

9. Nickel Prevent anaemia14, Strengthens bone, Hyperglycaemia, low B.P, Depression, Liver
(200µg-1000µg) breakdown of Glucose, Development of disease, Anemia, Low stomach acid, Sinus
Nucleic á congestion.

10. Lead ↓ Performance of Nervous system,

Nephropathy, ↑B.P, Anemia, Kidney, Brain
damage15, Miscarriage, Death.
11. Vanadium Increasing Insulin sensitivity16, Body- Reduced growth and Impaired 6
(1.8mg/day) building, Improve Glucose Control Reproduction

12. Zinc Synthesis of Proteins, Metabolism of RNA ↓Growth and Wound healing, Sterility,
(4mg-40mg) DNA, Modulate brain excitability, Low sperm count, Arthritis, High B.P,
Regulates apoptosis Impotence, DM, Depression, Kidney
stones.

13. Sodium Regulates Blood Vol. B.P, Osmotic Fatigue, Depression, Mental Apathy,
(1500mg-23000mg) Equilibrium and pH, Neuron function17 Hypertension18, Gout, Kidney damage,
Nausea, Vomiting.

14. Potassium Maintain Physiology, B.P, Vascular Tone
(300mg-15,000mg) Propagation of Action Potential, Active and
Passive Pumping of Ions, GI Motility19,
Fluid and Electrolyte Balance
Asthma, Irregular Heart Beats20,
Palpitations, Hypertension, Chest Pain,
Paralysis, Muscle Spasm, Bladder
Weakness, Frequent Menstrual Cycle, Wt.
Gain, Impotence.
 7

INDUCTIVE COUPLED PLASMA/OPTICAL EMISSION SPECTROSCOPY

It is a multi-element analysis technique that will dissociate a sample into its constituent atoms and ions and exciting them
to a higher energy level. Cause them to emit light at a characteristic wavelength21.
 High temperature (7000–8000 k),
 High electron density (1014–1016 cm3)
 Appreciable degree of ionization for many elements
 Simultaneous multi element capability (over 70 elements)
 Low background emission, and relatively low chemical interference22
 High stability leading to excellent accuracy and precision,
 Excellent detection limits for most elements (0.1–100 ng ml)
 Wide linear dynamic range (ldr)
 Applicable to the refractory elements
 Cost effective analysis.
 8
PRINCIPLE
 9
INSTRUMENTATION23
• NEBULISER : Liquid-aerosol-plasma
• DETECTORS : Optical rotation-electrical signals (CCD)
• ICP-TORCH
• SPRAY CHAMBER : Removes large droplets from aerosol
• PUMPS : Pushes the sample solution through the
tubings (peristalysis)
 10

FLAME PHOTOMETRY
Flame photometry is a process where in emission of radiation by neutral atoms is measured. The neutral atoms are obtained by
introduction of sample into flame. Hence the name flame photometry. Since radiation is emitted it is also called as Flame
emission spectroscopy24.

WORKING PRINCIPLE APPLICATIONS

 11
INSTRUMENTATION25
• SOURCE OF FLAME: Burner that provides flame
• NEBULIZER AND MIXING CHAMBER: Transport the solution into flame at steady rate
• OPTICAL SYSTEM: 3 PARTS
(1) CONVEX MIRROR: Transmit light emitted from atom and focus it on to the lens.
(2) CONVEX LENS: Focus the light on a point called slit.
(3) FILTERS: Reflections passing through slit reaches the filter which isolates the wavelength to be measured from
other emissions. It acts as interference type of colour filter.
• PHOTO DETECTORS: Detects the emitted light and measures the intensity of emitted radiations by flame and converts to
electrical signals.
WAVELENGTHS AND COLOR OF FLAME OF SODIUM AND POTASSIUM

Name of the
Emitted wavelength range (nm) Observed color of the flame
element

Violet
K 766

Yellow
Na 589
 12
LITERATURE REVIEW
• Separation/preconcentration of trace amounts of Cr, Cu and Pb in environmental samples by magnetic solid-phase
extraction with Bismuthiol-II-immobilized magnetic nanoparticles and their determination by ICP-OES(Suleiman
et.al 2009).

• Solid phase extraction of trace amounts of Ag, Cd, Cu, and Zn in environmental samples using magnetic
nanoparticles coated by 3-(trimethoxysilyl)-1-propantiol and modified with 2-amino-5-mercapto-1,3,4-thiadiazole
and their determination by ICP-OES(Mashhadizadeh et.al 2011).

• Development of cloud point extraction for simultaneous extraction and determination of Gold and Palladium using
ICP-OES( Tavakoli et.al 2008).

• Determination of Cd, Co, Ni and Pb in biological samples by microcolumn packed with black stone (Pierre noire)
online coupled with ICP-OES(Suleiman et.al 2008).
 BLACK GRAPES 13
PLANT PROFILE Kingdom : Plantae MUSK MELON
BANANA Division : Magnoliophyta Kingdom : Plantae
Kingdom : Plantae Class : Magnoliopsida Division : tracheophyta
Division : Magnoliophyta Order : Vitales Class : Magnoliopsida
Class : Liliopsida Family : Vitaceae Order : Cucurbitales
Order : Zingiberales Genus : Vitis Family : Cucurbitaceae
Family : Musaceae Species : Vitis vinifera Genus : Cucumis
Genus : Musa CHEMICAL CONSTITUENTS: Species : C. melo
Species : M. acuminata Bioactive phenolic compounds such as CHEMICAL CONSTITUENTS:
CHEMICAL CONSTITUENTS: Resveratrol, Quercetin and Rutin. Excellent source of vitamin A (68%) and
Contains Potash, Soda, Lime, Magnesia, Anthocyanins-red and purple color of the Vitamin C (61%). Melons are 90% Water
Alumina, Chlorine, Sulfuric Anhydride, grapes and wines31. Acids-tartaric and and 9% Carbohydrates, with less than 1%
Silica and Carbon anhydride. High K malic. Glucose, Fructose. K is the most each of Protein and Fat.
content – a Banana contains about 450 mg important mineral. USES:
of Potassium. Vitamin B,C, Oxalic acid, USES: Strengthens The Immune System,
Sulphate, Starch, Tannin, Glycosides, Astringent, Anti-inflammatory, Detoxifying Cardiovascular Health, Prevents Cancer,
Phenolic compounds, Gum mucilage30. and Antisclerotic. to stop hemorrhages and Combats Stress, Eye Health
USES: minor bleedings. Influence the kidneys, Prevents Diabetic Nephropathy,
Demulcent, Nutrient, Cooling, Astringent, promoting urine flow. Anemia and Beneficial For Lungs, Treatment Of
Antiscorbutic, Antifebrile, Restorative, exhaustion. Useful in cases of small-pox, Insomnia, Treatment Of Menstrual
Emmenagogue, Cardialgic, Styptic, neuralgia and insomnia, Treats Arthritis, Problems, Aids In Weight Loss, Quit
Laxative. Because of its high Potassium Promotes Digestion, Hydrates And Smoking Sans Withdrawal Symptoms,
content, bananas are naturally slightly Rejuvenates Skin, Anti-Ageing Benefits, Beneficial For Pregnant Women.
radioactive, more than other fruits. Treatment Of Eczema.
 BEETROOT TOMATO 14
(Beta vulgaris) (Solanum lycopersicum)
Kingdom : Plantae Kingdom : Plantae
Division : Magnoliophyta Division : Tracheophyta
Class : Magnoliopsida Class : Magnoliopsida
Order : Caryophyllales Order : Solanales
Family : Chenopodiaceae Family : Solanaceae
Genus : Beta Genus : Solanum
Species : B. vulgaris Species : Solanum lycopersicum L.
CHEMICAL CONSTITUENTS AND USES: CHEMICAL CONSTITUENTS:
Garden beet is very low in calories (provide only 45 kcal/100 g), Low in any fat contents and have zero cholesterol levels. they
and contain zero cholesterol and small amount of fat. Its nutrition are an excellent sources of Antioxidants, Dietary Fiber,
benefits come particularly from Fiber, Vitamins, Minerals, and Minerals, and Vitamins. Lycopene, a flavonoid antioxidant, Zea-
unique plant derived anti-oxidants32. xanthin, good levels of Vitamin A, and flavonoid anti-oxidants
The root is rich source of phytochemical compound, glycine such as α and ß-carotenes, xanthins and lutein. Vitamins such as
betaine. Folates, Thiamin, Niacin, Riboflavin as well some essential
minerals like Iron, Ca, Mn and other trace elements.
Betaine has the property of lowering homocysteine levels within
USES:
the blood. Homocysteine, one of highly toxic metabolite,
Plant used to make medicine. It is used for preventing Cancer of
promotes platelet clot as well as atherosclerotic-plaque
the Breast, Bladder, Cervix, Colon and Rectum, Stomach, Lung,
formation, which, otherwise, can be harmful to blood vessels.
Ovaries, Pancreas, and Prostate. It is also used to prevent
High levels of homocysteine in the blood result in the
Diabetes, Diseases of the heart and blood vessels
development of coronary heart disease (CHD), stroke and
(cardiovascular disease), Cataracts, and Asthma. to treat high
peripheral vascular diseases. blood pressure, Osteoarthritis, the common Cold, Chills,and
 15

AIM AND OBJECTIVES

World Health Organization currently encourages, recommends and promotes traditional/herbal preparation
in National Health Programmers because of it’s easily availability at low cost and safety. Even though the
herbal preparations are safe, but some of the herbal preparations cause serious poisoning and toxic effect,
due to the preparations containing dangerous toxic drugs or metals. There should be frequent monitoring
of herbal preparations, containing toxic drugs or metals.
The main aim of the project will be achieved through the following objectives
• Preliminary phytochemical screening of the selected plant samples.
• Analysis of the metal in selected plant samples was performed by ICP-OES.
• Determination of concentration of metals by ICP-OES.
 16
METHOD AND METHODOLOGY
•

MATERIALS REQUIRED
• CHEMICALS REQUIRED : Ethanol,
Milli Q water,
Standard Reagent
(Bottles 1000ppm)
Conc. Nitric Acid.
• INSTRUMENTS REQUIRED : ICPOES ,
Heating Mantles,
Hot plate
• GLASSWARE REQUIRED : Beaker (50 ,100ml), Volumetric Flask (20, 25 , 50 & 100ML)
Glass Rods, Micro Pipettes, Pipettes (1,5,10ml), (500 ml)
Round Bottom Flask and Condenser.
• MISCELLANEOUS : Test tube stand, Test tube holders, Filter paper, Butter paper,
Spatula, Thermometers, Stands, Tissue paper, Zip pouches,
Markers, Gloves, Labels, Cotton swabs, siDinfectant etc.
COLLECTION AND AUTHENTICATION OF PLANTS 17
The fresh fruits material was collected in the month of Feb -2017 in local market in Madinaguda, Hyderabad.
PREPARATION OF ALCHOLIC EXTRACT
Ethanolic extract - HOT CONDENSATION process
fruits(200-400g) + Ethanol(300ml) in RBF
↓
Condensation for 8hrs on heating mantle
↓ filter
Filtrate
↓
Evaporated at room temperature upto 2/3rd
↓
Residue was obtained after 2-3 days
↓
EXTRACT
PHYTOCHEMICAL SCREENING
TEST FOR GLYCOSIDES
A portion of the extract was
hydrolysed with Hcl and the
hydrolysate was subjected to Legal’s
and Borntrager’s test to detect the
presence of different Glycosides.
LEGAL’S TEST: To the extract, 1ml
of Pyridine and few drops of Sodium
Nitropruside were added and it was
made alkaline with NaOH. Appearance
of pink to red colour
BORNTRAGER’S TEST: Extract
was treated with Chloroform and then
the layer was separated. To this equal
quantity of dilute Ammonia soln was
added. Ammonium layer acquires pink
showing the presence of Glycosides.
TEST FOR SAPONINS
FROTH TEST: Place 2ml of extract
in water in a test tube. Shake well,
stable froth (Foam)is formed. Hence
saponins are present.
TEST FOR ALKALOIDS TEST FOR CARBOHYDRATES 18
DRAGENDORFF’S REAGENT: Drug MOLISCH TEST:To the extract add few
extract when treated with Potassium Bismuth drops of alcoholic α-naphthol, then add few
Iodide Solution gives reddish brown ppt. drops of conc. H2SO4 through sides of the test
MAYER’S REAGENT: Drug extract when
tube, violet colour ring is appeared at the
treated with Potassium Mercuric Iodide
solution gives cream color ppt. junction.
TEST FOR TANNINS TEST FOR PROTEINS
To the extract few ml of Chromic acid was XANTHOPROTEIC TEST: To 5ml of
added. No ppt was found. extract add 1ml of con Nitric acid and boil.
Yellow ppt was obtained. After cooling add
TEST FOR FLAVONOIDS
SHINODA TEST: To the extract add few 40% NaOH solution. Orange colour was
Magnesium turnings and con HCl drop wise. obtained.
Pink scarlet, Crimson red or occasionally green TEST FOR PHYTOSTEROLS
to blue colour appears after few minutes.
SALKOWSKI TEST: To the extract add few
ZINC HYDROCHLORIDE TEST: To the
extract add a mixture of Zn dust and con HCl. drops of con H2SO4, red colour at the lower
Gives red colour after few min. layer indicate the presence of sterol and
yellow colour presence indicate Phytosterols
TEST FOR MUCILAGE
are present.
To the extract Ruthenium red solution is added,
pink colour is obtained. Hence mucilage is
present.
PROCEDURE FOR ESTIMATION OF METALS 19
PROCEDURE:
PREPARATION OF EXTRACT SAMPLE Sample and standard were prepared
1g of extract was weighed ↓
↓ Integration time of 6min
5ml of HNO3 and heated on hot plate ↓
↓organic fumes evaporates Standard conc. were selected
Add 10ml of water for acid digestion ↓
↓digestion upto 50% Instrument was calibrated using standard solution
Evaporation of digested sample ↓
↓filtration Sample solutions were allowed to flow
Filtrate + 50ml water and further dilution ↓
↓ Reports were obtained in ppm
SAMPLE

PREPARATION OF STANDARD
The standard reagent 1000ppm was purchased from The National
Institute of Standards and Technology (NIST) from Pune. From the
standard reagent bottles (1000ppm) there were furthered dilutions into
0.5, 1, 2 ppm was prepared.
Process of the work:
Blank (10 min) std(0.5ppm)
Blank: std(1ppm)
Blank: std(2 ppm)
Blank: extract
 20

PROCEDURE OF FLAME PHOTOMETRY

Sample and standard solution were prepared
↓
Flame photometer was turned on
↓
Set blank with the diluent used for sample and standard preparation
↓
Aspiration of standard was done and recorded
↓
A graph was plotted conc on x-axis and intensity on y-axis
↓
Unknown sample aspiration was done and recorded

CALCULATION:
 21
RESULTS AND DISCUSSION
WEIGHT TAKEN FOR ESTIMATION OF METALS

PERCENTAGE YIELD OF THE EXTRACT

S.No Name of The Plant Percentage Yield (%)
1 Musa acuminate 10.4%
2 Vitis vinifera 12.6%
3 Beta vulgaris 9.03%
4. Solanum lycopersicum 15.3%

5 Cucumis melo 11.8%

 PHYTOCHEMICAL SCREENING 22
S.No Name of The Plant Alk Carb Gly Tan Phyto Flav sapo Pro Muc Alk : Alkaloids
Carb : Carbohydrates
Gly : Glycosides
1 M. acuminate + + + + - - + + - Tan : Tannins
2 V. vinifera + + + + + + + + - Phyto : Phytosterol
Flav : Flavanoids
3 B. vulgaris - + + + - - + + - Sapo : Saponins
4. S. lycopersicum + + - + - + - - - Pro : Proteins,
Muci : Mucilage.
5 C. melo + + - + - + + + -
The above table indicates the presence (+) or absence (-) of phytochemicals
 23

ESTIMATION OF METALS BY USING ICP-OES

NAME OF METAL ANALYSIS (ppm)
EXTRACT

As Ca Cd Cr Co Cu Fe Mn Ni Pb Vn Zn Na K
Musa acuminate -- 472.02 0.32 2.82 0.48 4.73 61.19 12.08 2.36 0.60 0.23 12.53 0.53% 0.45%

Vitis vinifera -- 657.16 0.40 4.26 0.49 5.84 37.24 7.45 1.63 0.47 0.17 14.71 0.16% 0.58%

Beta vulgaris 0.99 824.28 0.42 2.96 0.43 3.17 30.69 5.38 1.57 0.73 -- 17.88 0.15% 0.52%

Solanum -- 370.70 4.07 3.06 0.39 6.57 16.61 8.92 2.41 -- -- 13.83 0.08% 0.53%
lycopersicum

Cucumis melo 1.12 649.73 0.42 3.48 0.40 2.27 31.73 4.96 1.54 0.54 -- 6.20 0.04% 0.38%
24
 25
CONCLUSION:
• ICP-OES is a highly rated multi purpose analytical technique.
• Can be employed to determine over 70 different elements in solution or directly in solid samples.
• Wide applications in the field of Pharmacology, Biophysics and Toxicology.
• In our present study hydroalcoholic extracts of naturally occuring fruits were prepared followed by
phytochemical screening.
• This study suggest that various chemical constituents such as Flavanoids, Alkaloids, Carbohydrates, Tannins,
Proteins possesss activity against many health disorders.
• Each metal have its own significance to act as a micronutrients to human beings upto certain levels. Beyond
this level the metal is toxic and causes health risk. Hence it is essential to control the metal contents which are
exceeded.
 26
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