water purification revised.pptx

“ Development, Characterization and
Evaluation of poly herbal water
purifying sachet”
Supervised by: Dr. Deepti Jain
Co- Supervised by: Mr. Ram Singh Bisnoi
Presented By: Ankita Raikwar
0001PY17MP18
School of Pharmaceutical Sciences, Rajiv Gandhi Proudyogiki Vishwavidhalaya, Bhopal

CONTENT
 Introduction
i. Importance of water
ii. Types of impurities
iii. Test for impurity
iv. Limitation of RO water purifier
v. Advantages of herbs as water purifier
 Review of literature
i. Objective
ii. Plan of work
iii. Plant profile
 Materials and method
i. Collection and authentication of plants parts
ii. Extraction of phytochemical constituents of plants

iii. Determination of percentage yield of all extract
iv. Physiochemical characterization and preliminary qualitative phytochemical investigation
v. Isolation and qualitative identification characterization of compound
vi. Microbiological study
vii. Formulation of water purifying sachet
viii. Evaluation of water purifying sachet
 Result and discussion
i. Phytochemical screening
ii. FTIR spectroscopic fingerprint of different extracts
iii. Limit test for heavy metals of water sample
iv. Antibacterial activity of plants extract
v. Evaluation of purified water as per ISI standard specification
 Summary and conclusion
 References

WATER
 Good quality (potable) drinking water is free from disease-causing organisms, harmful chemical
substances and radioactive matter, tastes good, is aesthetically appealing and is free from
objectionable color or odor.
 It should be emphasized that there is a difference between "pure water" and "safe drinking water".
 Pure water, often defined as water containing no minerals or chemicals, does not exist naturally in
the environment.
 Safe drinking water, on the other hand, may retain naturally occurring minerals and chemicals such
as calcium, potassium, sodium or fluoride which are actually beneficial to human health.

COMMON IMPURITIES IN WATER
 Types of Impurity:
1. Biological content : algae, protozoa, bacteria, virus, microbes,
parasite, cysts(Eggs), biological contaminants
2. Chemical content: hardness (calcium + magnesium), chlorides,
sodium, organic mixes, metals(press and forth),
supplements(nitrogen and phosphorus)
3. Physical content: turbidity, shading, scent, dust, fine sand, clay,
etc.

TEST FOR IMPURITY
A.Test for heavy metals
Table no:- 1 Test for heavy metals
S. No. Class Test
1. Qualitative Limit test
2. Quantitative Atomic absorption Spectroscopy
Flame Photometry
Fluorimetry

B. Test for microbe
1.Presumptive test for coliform group of bacteria.
2.Water analysis for total bacterial population by disc diffusion method against standard ,blank ,ethanol
,herbal extract
3.Water analysis for total bacterial population by disc diffusion method against various dilutions of
herbal extract.
C. ISI specification for drinking water.

RO WATER PURIFIER
Limitations:
1. Removes essential minerals (Fe, Mg, Ca, Na).
2. Not kill bacteria, viruses.
3. Water taste altered.
4. More time to purify.
5. Water wastage.
6. Expensive.
7. RO membrane breakage.
8. Not mobile.
9. Requirement of electricity.

HERBS AS WATER PURIFIER
 ADVANTAGES
 Essential minerals remain.
 Antibacterial, Antifungal, Antiviral…..
 No wastage of water
 Cheap as compared to RO purifier.
 Mobile.
Soaked

 Khalil Noha et al. (2017) showed bactericidal effect of Coriander oil. In this study they proved
antimicrobial potential of coriander oil having potent bactericidal activity against E. coli and
Bordetella bronchiseptica and S.aureus. They found moderate bactericidal effect in all
bacterias.They also purpose to formulated dosage form to treat UTI caused by E. coli.
 Mohammad A. Alzohairy et al (2016) justified that Neem and its ingredients have therapeutics
implication and have been traditionally used .It is also reported that Neem plays pivotal role in
prevention of various diseases.
 Kumar Manoj et al (2012) stated investigation on various water quality parameter, chlorides,
dissolved oxygen, total iron, nitrate, water temperature, pH, total phosphorous, fecal coliform
bacteria, and adverse effect of these parameters on human being.
 Kulshreshtha A et al (2015) surveyed various areas of U P that are under the threat of heavy metals
and found content of heavy metals content above the range of parts per billion (ppb).They used
Inductive coupled plasma mass spectroscopy for the analysis of heavy metals. Results concluded that
concentration of metals exceeded WHO permissible limit for drinking water. Therefore
concentration of heavy metals needs to be controlled to control diseases that are linked with
utilization of heavy metals.

 Kaur S et al (2014)- Studied anti-oxidant activity and total phenolic content of alcoholic extracts
from seven medicinal plants (Asparagus racemosus, Ocimum sanctum, Cassia fistula, Piper betel,
Citrus aurantifolia, Catharanthus roseus, and Polyalthia longifolia).According to the experiment
highest antioxidant activity was demonstrated by Citrus auantifolia (87.05%) followed by Ocimum
sanctum (81.80%) and Catharanthus roseus (71.4%). The highest tannin content was found to be in
Catharanthus roseus (7.14%) while in case of anthocyanin content the highest value was found to be
in Polyalthia longifolia (0.65mg/l). As far as antimicrobial activity is concerned, Ocimum sanctum
and Citrus aurantifolia were found to be most potent against Escherichia coli and Staphylococcus
aureus whereas Piper betel showed no effect. The minimum inhibitory concentration against E. coli
in case of Ocimum sanctum is 10% and against S. aureus it is 20%.
 Dubey Rashmi et al (2015) investigated various chemical composition of Tulsi, isolated and
identified the compounds present in Tulsi herb by TLC and mass spectroscopy. They justified that
Tulsi reduces stress, enhances stamina and endurance, increases the body's efficient use of oxygen,
boosts the immune system, reduces inflammation, protects against radiation damage, lesions aging
factors, and supports the heart, lungs, and liver. Compounds present in Tulsi have antibiotic,
antiviral, and antifungal properties. It helps the body and mind to adapt and cope with a wide range
of physical, emotional, chemical, and infectious stresses.

 Sreelakshmi C.D (2017) justified treatment of wastewater from various industries etc. which
explained adverse percentage release of heavy metals like Cr,Pb, Fe , Cd, Ni, Cu etc. It can be
treated by various conventional methods like physicochemical methods, chemical precipitation,
coagulation and flocculation, electrochemical treatments, ion exchange, membrane filtration, electro
dialysis etc. Use of natural adsorbents are more eco-friendly when compared to other conventional
methods. In this investigatory work, Ocimum sanctum has been selected for the removal of heavy
metals like iron and lead, as an adsorbent. Stock metal solutions of iron and lead has been prepared
as samples of wastewater and confirmed that neutralization methods must be adopted for iron
solution. Color produced due to addition of Tulsi leaves poses a problem, it can be concluded that
Ocimum sanctum is more efficient in removing lead than iron.
 Das A et al (2018) their work incorporates planning, examining and assessing of antibacterial
strength of phytochemicals from Syzygium aromaticum (Clove) .Their work incorporates extraction
of S. aromaticum and its characterization utilizing TLC,GC–MS, and FT-IR. .Concentrate was
acquired by hydro distillation. Antibacterial activity was examined against various convergence of
clove extracts (25%, half, and 75%) against various pathogens, for example, Escherichia coli,
Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, Klebsiella oxytoca, and
Staphylococcus epidermidis.It was concluded from their work that clove concentrate utilizing the
natural dissolvable dichloromethane has compelling antibacterial action.


 Subapriya R et al (2005)-He reviewed that Azadirachta indica (Neem), become a cynosure of
modern medicine. More than 140 compounds have been isolated from neem.Every part of the Neem
have been used traditionally. Because of their relative safety and efficacy medicinal plants are used
worldwide for their phytochemical constituents. Most of the pharmacological properties of Neem
leaf have been reported only with crude extracts. Extensive investigations on the metabolism, tissue
distribution, pharmacokinetics, toxicity and the molecular mechanisms of chemo protection are
necessary for the development of modern drugs from Neem leaf.
 Elbagermi MA et al (2017) worked on determination of the amount of some heavy metals (Cu, Cr,
Ni, Cd, Mn, Fe,Mg and Pb) present in commercial brand herbal tea samples purchased from local
markets in by atomic absorption spectroscopy. The concentration of Cu, Cr, Ni, Cd, Mn, Fe, Mg and
Pb in all the tea leaf samples concluded out to be 5.141 to 17.1, 0.890 to 3.4, 0.0833 to 2.349, 0.035
to 0.38, 32.01to 89.46, 79.01-167, 91.98 to 213.83 and 0.463 to 0.901 mg g-1, respectively and
therefore data can be arranged in the following order, Mg> Fe> Mn>Cu >Cr> Ni> Pb> Cd. And
therefore result was concluded that concentrations of all the toxic elements tested in the investigated
herbal plants were found below the permitted levels specified by the international regulatory
standards for the medicinal plants.

 Mandal Shayamapa et al (2015) concluded that, C. sativum L., plants provide two types of herbal
raw materials-fruits and leaves, in which main activity is reported by essential oil. Essential oil and
extracts of the plant possess antibacterial, antifungal and anti-oxidative activities. They concluded
that essential oil plays a great role in maintaining the shelf-life of foods by preventing their spoilage.
 Ayoola GA et al (2008) performed steam distillation of the dry flower buds of Syzigium aromaticum
(Clove) and yielded 7% (w/w) of the pure oil. Their GC-MS analysis revealed presence of eugenol,
caryophyllene, eugenol acetate and alpha-humelene . The antimicrobial sensitivity of the volatile oil
against some Gram-negative bacteria(E. coli,Klebsiella pneumoniae, S. paratyphi,Citrobacter spp.
and Enterobacter cloacae), a Gram-positive bacterium (S. aureus), and fungus (Candida albicans)
which showed a broad spectrum of activity.MIC was determined for each organism as 2.4, 1.6, 0.27,
0.016, 0.23, 1.63, 0.73 and 0.067 mg/ml for S.aureus, E. cloacae, S. paratyphi, K. pneumoniae, E.
coli, E. coli,Citrobacter spp. and C. albicans, respectively. They also investigated antioxidant
screening of clove oil with 2,2-diphenylpicryl-hydrazyl radical (DPPH) was positive, indicating the
presence of free radical scavenging molecules which can be attributed to the presence of eugenol, a
phenolic compound.

 Yevate Ashwini et al (2017) worked on water purification process of removing undesirable physical,
chemicals, biologically contaminants from water. Water purification system proposed by him mainly
deals with purification of drinking water at reasonable cost with high reliability to the rural families
in remote area. Proposed system consists of combination of natural substances (Tulsi & Neem).They
also proposed effective method to remove fluoride from water. Method proved to be portable, cost
effective, user complaint and energy efficient which will be self-sufficient to meet the drinking water
needs.
 Biemer JJ (1973)-They discovered antimicrobial susceptibility by the disc diffusion method with
emphasis on the need for standardization of methodology. The Kirby-Bauer method is currently
widely used by clinical laboratories and approved by WHO. It is relatively simple from a
technological standpoint and accurate and reproducible so long as the methodology is followed with
care.
 Joshi B et al (2011)- worked on ethanol extract of 4 medicinal plants and studied in vitro
antibacterial assay against E. coli,S. typhi,S. paratyphi, S. aureus,Klebsiella pneumoniae,P.
aeruginosa by cup diffusion method. Among 4, Clove was most effective against S. typhi but all the
plants were ineffective against E. coli and K. pneumonia.Minimum Bactericidal Concentration
(MBC) value of 5 mg/l was obtained with Azadirachta indica against S. typhi. K. pneumoniae. They
performed qualitative phytochemical analysis for detection of alkaloids, glycosides, terpenoids,
steroids, flavonoids, tannins and reducing sugars. They also performed TLC using solvent system
chloroform, methanol and water (10:10:3)for the analysis of lipid present in plant extract.

 Aim of study
The aim of study is to formulate and evaluate a formulation that has herbs with application of anti-
microbial activity as well as can remove harmful heavy water from drinking water.
 Objective of study
1.To provide a water purification system based on natural phenomenon to purify water with minimum
side effects.
2.To provide a cost effective system to purify water to be effectively used in rural areas where there is
scarcity of good quality of water.
3.To make the system potable and user friendly.
4.To design a system in which essential minerals remain.
5.To design a formulation that with advantage to purify water it also lowers risk for illness, also boost
your mood at the same time.
 Expected outcomes
To design a formulation which has anti-microbial and as well as heavy metals removal property which is
portable at the same time.

 Rationale for selection of herbs:
In present work I have selected herbs (Tulsi, Clove, Neem, Moringa, coriander) which are effective
purification herb. The aim of study is to improve purification of water with elimination of microbes and
heavy metals and with a benefit of negligible amount of side effects.
 Rationale for selection of formulation:
Formulation of selection of water purifying bags as formulation is to benefit us with portability of
formulation that is a great disadvantage with RO system. With an objective of purification of water it is
going to be cost effective as well.

 Selection of plants and there parts
Coriander-leaves
Neem-leaves
Clove-buds
Tulsi-leaves
 Collection of selective parts from plants
 Extraction
Soxhlation with the help of soxhlet apparatus
 Evaporation of solvent
Rotatory evaporator
 Characterization by IR
 Microbiological study
Comparative study of standard ,blank ,ethanol ,drug extract
Concentration wise study of herbal extract

 Heavy metals study
 ISI water specifications
pH
Turbidity
E. coli
TDS(total dissolved solids)
Taste
Color
Odor
Heavy metals

1. NEEM (Azadiracta indica)
Fig - 1 Neem leaves
Taxonomical classification
Kingdom: Plantae
Division: Angiosperms
Class: Eudicots
Sub class: Rosids
Order: Sapindales
Family: Malieceae
Genus: Azadirachta
Species: A. indica

 Neem is a evergreen tree , and is native to the Indian subcontinent, i.e. India, Nepal, Pakistan,
Bangladesh, Sri Lanka, and Maldives. It is typically grown in tropical and semi-tropical regions. Its
fruits and seeds are the source of Neem oil.
 Macroscopic description- It is a fast-growing tree with 20–23 m tall and trunk is straight and has a
diameter around 4-5 ft. The leaves are compound, imparipinnate, with each comprising 5–15 leaflets.
Its fruits are green drupes which turn golden yellow on ripening in the months of June– August
 Chemical constituents- The most important active constituent is azadirachtin and other include
nimbidin, nimbidol, nimbin, quercetin, polyphenol.
 Pharmacology-Neem plays role as free radical scavenging properties due to rich source of
antioxidant. Azadirachtin and nimbolide showed concentration-dependent antiradical scavenging
activity and reductive potential in the following order: nimbolide > azadirachtin > ascorbate.It also
manages cancer by the regulating cell signaling pathways. It also plays role as anti-inflammatory via
regulation of pro inflammatory enzyme activities including cyclooxygenase (COX), and
lipoxygenase (LOX) enzyme

2. TULSI (Ocimum sanctum)
Fig - 2 Tulsi leaves
Kingdom: Plantae
Class: Eudicots
Sub class: Asterids
Order: Lamiales
Family: Lamiaceae
Genus: Ocimum
Species: Ocimum sanactum

 Ocimum sanctum (Tulasi) is a medium sized plant found in most of the regions of India, is an
aromatic perennial plant in the family Lamiaceae. It is native to the Indian subcontinent and
Southeast Asian tropics.Tulsi is cultivated for religious and traditional medicine purposes, and for its
essential oil.
 Macroscopic description- It is an erect, branched plant and its height is about 30-60 cm when
mature. The plant is acrid and bitter.Tulsi is aromatic and a branched herb covered with minute fine
hairs. Leaves are aromatic because they contain scented oil in them and they are simple, elliptic,
oblong, and obtuse with serrate or dental margins. Flowers are 5 mm long, with a calyx tube bearded
outside the base. The fruit is small with yellow to reddish colored seeds.
 Chemical constituents- The leaves contain a high content of essential oils. The extraction of fresh
leaves and stem yielded phenolic compounds like Eugenol, iso eugenol. Vitamin A and Vitamin C
are also found in this herb which stimulates antibody production up to 20% .
 Pharmacology- Tulsi is useful against stress; it enhances stamina and increases efficient use of
oxygen by body; strengthens immune system; reduces inflammation; protects from radiation;
reduces aging; supports the lungs, liver and heart; it exhibits antibiotic, antiviral and antifungal,
antioxidant properties. It cure cold, cough, headache, flu, asthma, fever, colic pain, bronchitis,
hepatic diseases, as an antidote, flatulence headaches, fatigue, skin diseases, wound, insomnia,
arthritis, influenza, digestive disorders, night blindness, diarrhea.

3. CLOVE (Syzygium aromaticum)
Fig - 3 Clove buds
Kingdom: Plantae
Class: Eudicots
Sub class: Rosids
Order: Myrtales
Family: Myrtaceae
Genus: Syzygium
Species: S aromaticum

 Cloves are the aromatic flower buds of a tree in the family Myrtaceae, Syzygium aromaticum. They
are native to the Maluku Islands in Indonesia, and commonly used as a spice. Cloves are available
throughout the year due to different harvest seasons.
 Othernameslavangaa.devakusumum(Sanskrit),lavang(Bengal,gujrat),laung(hindi),lavanga(kannada)k
arayampu,krambu(malyalam),lavang(Marathi),kirampu,lavangam(tamil),lavangamulu (telugu).
 Macroscopic character- flower buds are reddish brown,16-21 mm long.Hypanthium flattened-
cylindrical or some what 4 sided and surrounded by 4 spreading,thick acute sepals.The head consists
of 4 membraneous, imbricated petals and numerous incureved stamens around a large style. A ovary
situated in upper part of hypanthium,The lower part of hypanthium is solid, though rather spongy
near the cortex. Ovary bilocular each ovary contains about 20 ovules attached to axile placenta.
Odour strong, spicy and aromatic. Taste-agreeable warm and aromatic.
 Chemical constituents-phenol, eugenol, eugenol acetate.
 Pharmacology-Cloves have a positive effect on healing of stomach ulcers,anti-carcinogens, anti-
oxidative; possess growth inhibitory activity against oral pathogens, carminative, stomachic.

4.CILANTRO (Coriandrum sativum)
Fig no:- 4 Coriander leaves
Kingdom: Plantae
Class: Eudicots
Sub class: Asterids
Order: Apiales
Family: Apiaceae
Genus: Coriandrum
Species: C. sativum

 Coriander is an annual herb native of Mediterranean region and extensively cultivated throughout
India.in the family Apiaceae. It is also known as Chinese parsley, and the stems and leaves are
usually called cilantro in North America. All parts of the plant are edible, but the fresh leaves and the
dried seeds (as a spice) are the parts most traditionally used in cooking.
 Othernamesdhanyaka,danya(Sanskrit),dhane(Bengal),dhana,kothmir(gujrat),dahania,kottmir(hindi)k
othambribeeja(kannad),kothumpalari(malyalam),dhanna(Marathi),kothmalli(tamil),dhaniyalu(telugu
)
 Macroscopic description-Fruits consists of oval shaped cremocarp having 2 hemi spherical
mericarpsunited by their margins.Each pericarp has 5 wavy rather inconspicuous primary ridges
alternating with 4 more prominent secondry ridges. Its fruits are aromatic odor and spicy in taste.
 Chemical constituents-Major-essential oil ( ̴1%), including linalool (60-70%).Minor-monoterpene
hydrocarbonviz.ßpinene,αpinene,limonene,γterpinene,pcymene,citronellol,camphor,geraniol,coriand
rin,coriandriones,flavonoids,phenolic acids and sterols.
 Pharmacology-fruit extract of C. sativum inhibits mycelial growth of Pythium aphanidermatum, it is
strong fungitoxicity at very low concentration. Stomachic, spasmolytic and carminative,
hypolipidemic, preventive effect on lead deposition, insulin-releasing, microbicidal.

COLLECTION AND AUTHENTICATION OF
PLANTS PARTS
Collection of sample-Medicinal plants leaves (Tulsi, Neem, Coriander) and buds (Clove) used for the
experiment were collected from from RGPV botanical garden and local vendors according to their
availability and folk use and identified using literature.
Plants were sun dried under the shade and stored in zip lock bag. All chemicals taken were are of
chemical grade. Instruments and equipment were utilized from RGPV College Bhopal.
 Chemicals Required- Distilled water,methanol,ferric chloride, silica gel,methanol,chloroform
 Glasswares required-distillation assembly,petridish,beaker,measuring cylinder,spatula,glass
rod,iodine flask,TLC chamber,glass slides.
 Equipments used-rotatory evaporator,centrifuge,FTIR( shimadzu),autoclave,inoculation chamber

EXTRACTION OF PHYTOCHEMICAL
CONSTITUENT S OF PLANTS
Leaves(neem,tulsi,coriender)/Buds(clove) (10 gm)
↓(dried in shade)
Washed with distilled water
↓
Defatted with petroleum ether
↓
Macerated with 70% ethanol (soxhlet apparatus)(100ml)
↓ 12 hours
methanolic extract was filtered through whatman paper
↓
Filterate was concentrated at room temperature near sun
shaded place
↓
Dried extract was scratched and collected in ependroff tube
and stored in refrigerator.
Fig no:- 6 Successive extraction

MACERATION
 Maceration of herbs-10 g of powdered herb was macerated with 100 ml of 80% methanol for 24-48
hours.
 Extract obtained after filtration by muslin cloth was allowed to concentrate under rotatory
evaporator.
Fig no:- 7 Methanolic extract

PHYSIOCHEMICAL CHARACTERIZATION AND
PRELIMINARY QUALITATIVE PHYTOCHEMICAL
INVESTIGATION
 Phytochemical screening-Phytochemical subjective investigation incorporates appraisal of plant
extricates and ethanol watery answers for nearness of phytochemical constituents. It includes
extraction, screening and recognizable proof of restoratively dynamic substances found in plants. A
portion of the bioactive substances are flavonoids, alkaloids, carotenoids, tannin, cell reinforcements,
and phenolic mixes
1. Test for phenolic content
Chlorogenic acid test-Treat the test solution with aqueous ammonia and expose to air gradually.
2. Test for flavonoids
1 ml of extract in a test tube + few drops of dilute NaOH solution. An intense yellow color was appeared
in the test tube. It became colorless on addition of a few drops of dilute hydrochloric acid that indicated
the presence of flavonoids.

 Limit test for heavy metals
Table no:- 2 Procedure for limit test
TEST SAMPLE STANDARD COMPOUND
Solution is prepared as per the monograph and
25 ml is transferred in Nessler’s cylinder or
weigh specific amount of substance and
dissolve in 20 ml of water and add 5 ml of
dilute sodium hydroxide solution.
Take 2 ml of standard lead solution
Make up the volume to 50 ml with water Add 5 ml of dilute sodium hydroxide solution and
make up the volume to 50 ml with water
Add 5 drops of sodium sulphide solution Add 5 drops of sodium sulphide solution
Mix and set aside for 5 min Mix and set aside for 5 min
View downwards over a white surface View downwards over a white surface

S. No. PLANT ACTIVE CONSTITUENTS EFFECTIVE AGAINST
1 Tulsi
(Ocimum sanactum)
Krishna Tulsi (“phenolic
content”)
Vana Tulsi (anti-oxidant)
Eugenol
Microbes- Staphylococcus
aureus, E. coli, Pseudomonas
aeruginosa
Matter- Fluoride
2 Clove
(Eugenia caryophyllus)
“Eugenol”, Phenol Microbes- Staphylococcus
aureus, E.coli, Pseudomonas
aeruginosa, Streptococcus
pyrogens, Cyanobacteria,
Salmonella, Bacteriods fragilis,
Candida albicans
3 Cilantro
(Coriandrum sativum)
“Plantaricin CS”, linalool Metals-Pd , Ni, Ar, Hg, Cd
Microbes- Escherichia
coli, Bordetella bronchiseptica
and Staphylococcus aureus,E
4 Neem
(Azadiracta indica)
Nimbin, Polyphenol
compounds, Azadiractin
Nematodes, E. faecalis

Table no:- 1 Herbs profile with its active constituents, dose and application
S
No.
Herbs Active Constituents Dose Application (Effective againt)
1. Tulsi (Ocimum sanactum) phenolic content,anti-
oxidant(ß-carotene)
Eugenol
40μg/ml Microbes-staphlococcus aureus,E. coli,Pseudomonas
aeruginosa
Matter-flouride
2. Clove(Eugenia caryophyllus) Eugenol, terpene,thymol,
flavanol, phenol
30μg/ml Microbes-staphlococcus aureus, E. coli, Pseudomonas
aeruginosa,
Streptococcus pyrogens, Cynobacterium, Salmonella,
Bacteriods fragilis, Candida albicans
3. Neem (Azadiracta indica) Leaves-quercitin,
bsitosterol,
polyphenolic,flavanoids
30μg/ml Nematodes, E. faecalis
4. Cilantro(Coriandrum sativum) Plantaricin CS, linalool 40μg/ml Metals: Pd, Ni, Ar, Hg, Cd
Microbes: Escherichia coli, Bordetella bronchiseptica and
Staphylococcus aureus

8. Standard for Safe Drinking Water [The Bureau of Indian Standards (BIS)]
Table no:- 10 Standard for water testing parameters
Test parameter Acceptable limit Permissible limit
(In the absence of
alternate source of
water)
Limit after
treatment with
herbal water
pH value 6.5-8.5 No
relaxation
No relaxation 7.2
Turbidity 1 5 3
E.coli presence/absence Shall not be detectable in any 100ml sample Shall not be detectable
in any 100ml sample
Absent
TDS (Total dissolved
solids) mg/l, max
500 2000 820
Taste Agreeable Agreeable Herbal taste
Colour Transparent Transparent Slight yellowish
Odour Agreeable Agreeable Nil
Heavy metals(limit test) Brown Transparent Slight
yellowish(due to
herbs)

ISOLATION AND QUALITATIVE IDENTIFICATION
CHARACTERIZATION OF COMPOUND
1. TLC (Thin layer chromatography)
TLC technique deals with separation, identification and estimation of various components present in the
sample. It works on the principle of adsorption. Technique involves stationary phase which is fixed
(silica gel) and mobile phase which runs over stationary phase (liquid, gas).
Preparation of TLC plate:-Glass plate of 20*20 was coated with silica gel G grade and were allowed to
dry at room temperature on a flat surface for 5-10 min, then were allowed to dry at 1000 C for 20 min
and allowed to cool at room temperature.
Development of TLC chamber:-TLC chambers were taken and desired solvents were allowed to put in
chamber and chamber was covered for some time for saturation.
Preparative TLC:-TLC was performed on silica gel G coated plates by spotting the extract (Neem, Tulsi,
Clove, Coriander) over silica plates with thin capillary tubes. And then plates were allowed to rest under
mobile phase saturated chamber until mobile phase entire running over stationary phase. Finally they
were taken out from chamber and visualized and examined for the spots developed.
Stationary phase : Prepared silica gel G coated plates

Mobile phase :chloroform: methanol: water (5:4:1)
Chamber saturation time: 15 min.
Development technique : Ascending technique
Sample : ethanol extract
Detection : visibly
2. IR (Infrared spectroscopy) Spectroscopy
IR Spectroscopy deals with infrared region of electromagnetic spectrum, i.e. light having a longer
wavelength and a lower frequency than visible light .It generally refers to analysis of interaction of a
molecule with infrared light .It can be analyzed in three ways: by measuring reflection, emission, and
absorption .The major use of infrared spectroscopy is to determine the functional groups of molecule.
IR spectroscopy detects frequencies of infrared light that are absorbed by a molecule .Molecules tend to
absorb these specific frequencies of light since they correspond to the vibration of bonds in the
molecule.
Principle-The IR spectroscopy theory utilizes the concept that molecule tend to absorb specific
frequencies of light that are characteristic of corresponding structure of the molecules. The energies are
reliant on the shape of the molecular surfaces ,the associated vibronic coupling, and the mass
corresponding of the atoms .For instance ,the molecule can absorb the energy continued in the incident
light and the result is a faster rotation or a more pronounced vibration.

MICROBIOLOGICAL STUDY
 Microbiological study
1. Preparation of dilutions
Plant extracts(1mg) was diluted in distilled water (10 ml) to prepare stock solution,further 1ml from the
above solution was taken and diluted with distilled water(10) to prepare sub-stock.From this sub-stock
solution prepare further serial dilutions i.e. 10μg/ml ,20 μg/ml ,30 μg/ml ,40 μg/ml.
Sterilized disc were then prepared with Whatman filter paper which were cut circular in shape,disc were
washed with ethanol then discs were dried in 37°C incubator for 15 minutes and then placed under UV
aseptic chamber for further sterilisation.
2. Sterilisation of UV chamber and incubator .
UV chamber was sterilized using 80% ethyl alcohol swab running around the glass walls .Incubator was
steriluzed using 80% ethyl alcohol swab cleaning parallely along each end.
3. Bacteria Culture
Bacteria to be used for the test were obtained from various contaminated water bodies as well as tap
water i.e. water was taken as baterial sample.

4. Disc Diffusion Method
Disc diffusion method for antimicrobial susceptibility testing was carried out according to the standard
method by Bauer et al. (1966) to assess the presence of antibacterial activities of the plant extracts.
Bacteria culture has been used to lawn Muller Hinton agar platesevenly using a sterile bent capillary
prepared. The plates were dried for 15 minutes and then used for the sensitivity test. The discs which
had been impregnated with a series of plant extracts were placed on the MuellerHinton agar surface.
Each test plate comprises of four discs. One positive control, which is a standard commercial antibiotic
disc(ciprofloxacin), one negative control(blank disc), one disc with 70% ethyl alcohol and fourth one
with herbal extract.Each plate consists of four discs placed about equidistance to each other. The plates
were then incubated at 37°C for 18 to 24 hours . After the incubation, the plates were examined for
inhibition zone. The inhibition zone were then measured using calipers and recorded. The test were
repeated three times to ensure reliability.

FORMULATION OF WATER PURIFYING SACHET
Fig no:- 15 Water purifying bag
 Tetrahedron shaped
Material: Poly lactide (PLA), a bio-plastic, filter paper, food grade plastic, silk cloth.
Water Purifying Bags: little, permeable, fixed sack or parcel containing dried plant material or active
constituent extracts, which is drenched in bubbling water to make an infusion.

1 Percentage yield of different extract:-
Table no:- 3 Percentage yield
2. Test for phenolic content
Tulsi-present(green brown)
Clove-present(blackish green)
Coriander-no distinctive color change.
Neem-present(green blue)
3. Test for flavonoids
i. Tulsi-present (brown)
ii. Clove-present (yellowish brown)
iii. Coriander-present (dark yellow).
iv. Neem-present (intense yellow).
S. No. Extract % Yield
1. Clove 8.9%
2. Neem 7.2%
3. Tulsi 6.9%
4. Coriander 7.5%

4. Test for heavy metals
Fig no:- 18 limit test standard, test and herb
Intensity of test solution when compared to test sample was low, while on comparing additional herbal
water altogether with standard and herbal water sample found out to be low than standard therefore it
was able to pass heavy metal limit test for water.

5. Characterization by TLC
Rf value=Distance travelled by solute/Distance travelled by solvent
1. Tulsi
Fig no:- 19 TLC plate for Tulsi
Rf value of extracted eugenol=3.1/5
=0.62
Rf value of standard eugenol = 0.65

2. Clove
Fig no:- 20 TLC plate for Clove
Rf value of extracted eugenol=3.2/5
=0.64
Rf value of standard eugenol = 0.65
Reddish brown spot -0.37(eugenol)
Dark violet spot-0.94(ß-caryophyllene)

3. Neem
Fig no:- 21 TLC plate for Neem
Rf value of extracted azadiractin=2.97/5.2
=0.57
Rf value of standard azadiractin =0.55

4. Coriander
Fig no:- 22 TLC plate for Coriander
Rf value of extracted linalool =1.55/5
=0.31
Rf value of standard linalool =0.35

6. Characterization by IR
Most convenient technique for identification of functional group and compound .Comparison of
standard and extract is shown in given spectra:
1. Tulsi
Fig no:- 23 IR spectra of tulsi
Table 4.4 Observation table of interpretation
of IR spectra-TULSI
Table no:- 4 IR spectra interpretation tulsi
Conclusion-IR spectra of Tulsi extract shows presence of
alkyl (1458.18), methoxy (2857.86),
benzene(1458.18),hydoxy group (3437.15),aromatic ring
(1440.83), and primary alcohol at (1049.28) which
confirms the presence of eugenol,eicosanoic acid,
cyclohexane and nitrogen derived aromatic ring.
S. No. Peak Functional
group
1. 1458.18 CH2
2. 2875.86 OCH3
3. 1458.18 Benzene
4. 3437.15 OH
5. 1440.83 Aromatic ring
6. 1049.28 Primary alcohol

2. Clove
Fig no:- 24 IR spectra of clove
Table no:- 5 IR spectra interpretation clove
Conclusion--IR spectra of Clove extract shows
presence of ammonia (3066.82), methoxy group
(2850.79),alkenes(2931.8),hydroxyl group
(1269.16),aromatic ring(1460.11) and phenolic
group (1197.79) which confirms the presence of
eugenol which is potent antimicrobial and heavy
metals removing property.
S. No. Peak Functional group
1. NH3 3066.82
2. OCH3 2850.79
3. C=C 2931.8
4. OH 1269.16
5. Aromatic ring 1460.11
6. CH3 1460.11
7. Phenol 1197.79

3. Neem
Fig no:- 25 IR spectra of neem Table no:- 6 IR spectra interpretation neem
 Conclusion--IR spectra of Neem extract shows
presence of carboxyl (2873.94),alkyl
group(1375.25),benzene(1043.49),carboxylic acid
(1724.36) and phenolic group at (1219.01)
confirms presence of azadiractin and querecitin
which are antimicrobial agents with heavy metal
removal property.
1. C=O 2873.94
2. CH3 1375.25
3. Benzene 1043.49
4. CH3COOH 1724.36
5. Phenol 1219.01

4. Coriander
Fig no:- 26 IR spectra of coriander
Table no:- 7 IR spectra interpretation coriander
Conclusion--IR spectra of Coriander extract shows
presence of hydroxyl group(1259.52),amine
group(1029.99),primary alcohol(1049.28), sulphur
(1338.6),alkyl group(2872.01) and alkene(630) that
confirms presence of Plantaricin CS which is the
main agent of antimicrobial activity with heavy
metal removal property.
1. 1259.52 OH
2. 1029.99 NH2
3. 1049.28 Primary alcohol
4. 1338.6 S
5. 2872.01 CH3
6. 630 C=C

 Invitro antibacterial activity of plant extract
1. Coriander
Fig no:- 8 Culture plate showing comparative study of blank, ethanol, standard, coriander disc

2.Tulsi
Fig no:- 9 Culture plate showing comparative study of blank, ethanol, standard, tulsi disc

3.Clove
Fig no:- 9 Culture plate showing comparative study of blank, ethanol, standard, clove disc

4. Neem
Fig no:- 10 Culture plate showing comparative study of blank, ethanol, standard, neem disc

7. In vitro anti-bacterial activity of plant extract
1. Culture plate showing comparative study of blank, ethanol, standard and plant extract
Table no:- 8 Culture plate data showing comparative study of blank, ethanol, standard and plant
extract
Conclusion-According to the present culture plate study it is observed that blank shows negligible result,
while inhibition is observed in ethanol disc ,standard (ciprofloxacin) shows more inhibition in
comparison to herbal extract overall ,while the result obtained from herbal extract is pretty much similar
to standard drug. Which gives conclusion that Tulsi has more potent than standard, Neem, Coriander and
Clove extract is less potent than standard.
Herbs Blank
Zone of inhibition
(mm)
Ethanol
Zone of inhibition
(mm)
Standard
Zone of inhibition
(mm)
Herbal extract Zone
of inhibition
(mm)
Tulsi 0 2 22 25
Neem 0 5 25 22
Clove 0 0 25 22
Coriander 0 5 23 18

 In vitro antibacterial activity of plant extract(Concentration wise study of herbal extract)
1. Clove
Fig no:- 11 Culture plate showing comparative study of concentration (10μg/ml, 20μg/ml, 30μg/ml,
40μg/ml) clove extract disc

2. Coriander
40μg/ml) coriander extract disc

3. Tulsi
40μg/ml) tulsi extract disc

2. Culture plate showing comparative study of concentration (10μg/ml, 20 μg/ml, 30 μg/ml, 40 μg/ml) plant
extract.
Table no:- 9 Culture plate data showing comparative study of concentration of plant extract
Conclusion-According to the various concentration wise study of herbal extract it was observed that at 10μg/ml
conc. clove is potent with 2mm zone of inhibition while Tulsi, Neem and Coriander have negligible effect ,at 20
μg/ml conc. Clove is more potent that rest of 3 drugs whereas Tulsi and Neem have no effect,at 30 μg/ml Tulsi
and coriander extract have maximum potency of 20mm whereas Neem and clove have 17mm,at a conc. 40
μg/ml potency of every drug is maximum as compared to previous and is maximum in case of Tulsi.
Plant material(crude
extract)
Zone of inhibition
(mm)
Zone of inhibition
(mm)
Zone of inhibition
(mm)
Zone of inhibition
(mm)
10μg/ml 20 μg/ml 30 μg/ml 40 μg/ml
Tulsi 0 0 20 22
Neem 0 0 17 19
Clove 2 12 17 18
Coriander 0 7 20 20

EVALUATION OF WATER PURIFYING SACHET
1. Microbiological test of mix
(neem+clove+coriander+tulsi)
Fig no:- 16 Culture plate showing mix extract
disc
2. Limit test
Fig no:- 17 limit test standard, test and herb

SUMMARY AND CONCLUSION
 Plants with aid of negligible side effects are richest source of chemical entity.
 Present study includes utilization of some medicinal plants after extraction and phytochemical
investigation.
 Ethanol extract obtained proved to be a positive result.
 Formulation obtained abides to antimicrobial and heavy metal removal property of water sample.
 Formulation is a cost effective and portable system.

water purification revised.pptx

water purification revised.pptx

Recommended

Recommended

More Related Content

Similar to water purification revised.pptx

Similar to water purification revised.pptx (20)

More from Ankita Raikwar

More from Ankita Raikwar (6)

Recently uploaded

Recently uploaded (20)

water purification revised.pptx