B.Tech Thesis

1
PROJECT REPORT ON
An attempt to develop Curcumin Nanoparticles using Calcium
Carbonate treatment: Preparation, Characterization and its
Antimicrobial Activity
Submitted in
Partial fulfilment of the requirements for
The Degree of Bachelor of Technology (Biotechnology)
Under the supervision of
Dr. Anil Kumar
Professor & Head
Department of Molecular Biology and Genetic Engineering
COLLEGE OF BASIC SCIENCES AND HUMANITIES
G. B. Pant University of Agriculture & Technology
Pantnagar – 263145 (U.K)
Submitted by
KAUSHALESH GUPTA KANIKA KALRA
I.D:- 40175 I.D:- 40177
VIJAY SINGH KASHIA
I.D:- 40176

2
ACKNOWLEDGEMENT
This is perhaps the easiest and the hardest part that I have to write in my report. It will be
simple to name all the people who have helped to get this done, but will be tough to thank
them enough.
We take immense pleasure in thanking Department of Molecular Biology and Genetic
Engineering, CBSH, Pantnagar for providing us the opportunity to carry out this project
work. We are sure this tenure will surely help us for lifetime and guide our future as we
have had great experiences while working for the project.
Firstly, we would like to express our heartfelt and sincere gratitude to our guide and
mentor, Dr. Anil Kumar, for giving us the opportunity to carry out our project under shade
of his knowledge. We wish to express our gratitude for his competent guidance,
supervision and intelligent attention to our work. We always felt comfortable going to him
if an experiment wasn’t working, and knew he could help us figure out something else to
try. The discussion session with him were very informative and helped us a lot in thinking
clearly about our work. We thank him for keeping us motivated and inspiring us at the low
tides.
We also want to thank Dr.Uma Melkania (Dean, CBSH, Pantnagar) for her continuous
guidance, never ending support, motivation and faith in our abilities.
Furthermore, we would also like to acknowledge with much appreciation the crucial role
of Dr.Manoj Singh, Miss Pallavi Shah and Miss Vishakha who guided us through this
project and helped us to assemble the parts and gave suggestion about the task. Sincere
thanks to all the faculty members, non- teaching and technical staff of the Department of
Molecular Biology and Genetic Engineering for their co-operation and assistance. Many
thanks go to all our teachers who gave full effort in guiding the team in achieving the
goals during the degree program
We have to appreciate the guidance given by our parents who were always there to teach
us important things in life.
Finally, we would like to owe our sincere thanks to our friends for their unconditional
love, blessings and total support and for always encouraging us to do our best .This project
would never have been possible without them.
Kaushalesh Gupta

3
CERTIFICATE
This is to certify that the project work entitled “An attempt to develop Curcumin
Nanoparticles using Calcium Carbonate treatment (Preparation,
Characterisation and Antimicrobial Activity)” submitted in partial fulfilment of the
requirements for the degree of Bachelor of Technology in Biotechnology, G. B. Pant
University of Agriculture and Technology, Pantnagar, is a genuine account of work
carried out by Kaushalesh Gupta, I.D. No. 40175, under my supervision.
Date: 17/06/2014 Dr. Anil Kumar
Place – Pantnagar Head and Professor

4
Declaration by the Candidate
This is to declare that this written submission in my project entitled “An attempt to
develop Curcumin Nanoparticles using Calcium Carbonate treatment (Preparation,
Characterisation and Antimicrobial Activity)” represents my work during the last year
of my B.Tech degree programme i.e. from Aug 2013- June 2014 at Department of
Molecular Biology & Genetic Engineering, CBSH under the supervision of Dr. Anil
Kumar.
I have adequately cited and referenced the original sources I also declare that I
have abided to all the principles of academic honesty and integrity and have not
misrepresented or fabricated or falsified any idea/data/fact/source in my submission.
I understand that any violation of the above will be a cause for disciplinary action
by the college and can also evoke panel action from the sources which have thus not been
properly cited or from whom proper permission has not been taken when needed.
Kaushalesh Gupta

5
CONTENTS
1. Introduction
2. Review of literature
3. Materials, Instrumentation and Methodology
4. Results and Discussions
5. Summary Conclusion
6. References

6
INTRODUCTION: Chapter 1
The dried and powdered rhizomes of Curcuma longa L., Zingiberaceae, commonly known
as turmeric, are used worldwide as a food-coloring agent. Several in vitro and in vivo
studies have confirmed that turmeric extracts have powerful biological activities, such as
anti inflammatory (Jurenka, 2009), antibacterial (De et al., 2009), antidepressant (Kulkarni
et al., 2009), antidiabetic (Wickenberg et al., 2010), antitumor (Wilken et al., 2011),
immunomodulatory (Rogers et al., 2010) and gastroprotective (Kim et al., 2005)
properties. In addition, it has been successfully used in the treatment of Alzheimer’s
disease (Ahmed et al., 2010) and cardiac disorders (Morimoto et al., 2010). Owing to its
antioxidant properties, turmeric has been widely accepted as one of the spices with the
highest antioxidant activity (Wojdyło et al., 2007).
The antioxidant activity of turmeric justifies its use in a broad range of
applications, including cosmetics (Thornfeldt, 2005), nutraceuticals (Aggarwal, 2010) and
phytomedicines (Aggarwal and Harikumar, 2009). These medicinal attributes can be
related to turmeric’s high content of curcuminoids, especially curcumin, which is
considered a chemical marker of this specie (Gupta et al., 2012).
The potential health benefits of curcumin are limited by its poor solubility, low
absorption from the gut, rapid metabolism and rapid systemic elimination. Some
Curcumin formulation(SuperBio-Curcumin®) absorbs up to seven times better than
conventional curcumin, thus represents the most cost-effective way to achieve greater
peak blood levels, twice as long as conventional supplements. Each 400 mg capsule of
Super Bio- Curcumin® is equivalent to 2,772 mg of a typical 95% curcumin extract 58.
To improve the bioavailability of curcumin, numerous approaches have been undertaken.
These approaches include:
i) The use of adjuvant like piperine that interferes with glucuronidation,
ii) The use of liposomal curcumin,
iii) Use of curcumin nanoparticles
iv) The use of curcumin phospholipid complex
v) The use of structural analogues of curcumin.
The use of structural analogues of curcumin has a rapid absorption with a peak
plasma half-life 59. The oral administration of the curcumin nanoparticles (poly lactic-co-

7
glycolic acid) enhanced the bioavailability up to 5.6-fold and had a longer half- life
compared with that of native curcumin. The improved oral bioavailability may be
associated with improved water solubility, higher release rate in the intestinal juice,
enhanced absorption by improved permeability.
Besides this curcumin has potential application in the field of nanotechnology.
Curcumin nanoparticles (less than 50nm in diameter) are highly effective in enhancing
cytotoxicity in treatment of breast cancer when compared with the conventional curcumin.
These also act as an anti-inflammatory and anticancer agent. In-vitro studies suggested
that the curcumin incorporated in nano fibres exhibited sustained release and maintain its
free radical scavenging ability. NanoDoxCurc (doxorubicin-curcumin composite
nanoparticle) have the ability to overcome multidrug resistance .In addition, it also showed
complete absence of cardiac toxicity in mice receiving high cumulative doses of
doxorubicin by reducing in tracellular oxidative stress whereas administration of free
doxorubicin and pegylated liposomal doxorubicin (Doxil®) results in attenuation of
cardiac function and cause hematological toxicities. Thus NanoDoxCurc overcomes both
multi drug resistance based doxorubicin chemo resistance and doxorubicin- induced cardio
toxicity and act as a potent anticancer agent. Curcumin loaded cellulose nanoparticles
(cellulose- CUR) formulation has potential use in prostate cancer therapy. Curcumin-
loaded magnetic nanoparticles demonstrated greater and sustained anti proliferative
activity in a dose- and time-dependent manner and also activate multiple signaling
pathways for provoking the anti-leukemic activity. Curcumin encapsulated- PLGA
nanoparticles were found to destroy amyloid aggregates, exhibit anti-oxidative property
and are non- cytotoxic. However, the encapsulation of the curcumin in PLGA does not
destroy its inherent properties and thus the PLGA-curcumin nanoparticles can be used as a
drug with multiple functions in treating Alzheimer’s disease. Nano curcumin enhance the
clinical efficacy by enabling aqueous dispersion. Nano curcumin had better dispersibility
and enhanced bioavailability in hydrophilic environment as compared to normal curcumin.
Considering the so much unexplored properties of the turmeric’s main active
constituent- curcumin; scientists are working enthusiastically for utilising the hidden
applicability of this component in human welfare. Regarding HPLC, the work is going on
in determining the presence of curcumin in rat plasma and in mouth ulcer poly herbal
formulation. While in case of synthesis and analysis of nano particles formed in turmeric,
there are various ways that scientists are referring towards. There is a green approach to
synthesize gold nano particles using turmeric rhizome extract, curcumin and curcumin

8
glycoside acting as reducing and stabilizing agents.
The frequency of life-threatening infections caused by pathogenic microorganisms
has increased worldwide, becoming an important cause of morbidity and mortality in
immuno compromised patients in developing countries. Although a large number of
antimicrobial agents have been discovered, pathogenic microorganisms are constantly
developing resistance to these agents. Antibiotics are sometimes associated with side
effects whereas there are some advantages of using antimicrobial compounds of medicinal
plants. The later has fewer side effects, better patient tolerance, relatively less expensive,
acceptance due to long history of use and being renewable in nature. Antibacterial
constituents of medicinal plants and their use for the treatment of microbial infections as
possible alternatives to synthetic drugs to which many infectious microorganisms have
become resistant seem to be very much promising. Over the past 20 years, there has been a
lot of interest in the investigation of natural materials as sources of new antibacterial
agents. Different extracts from medicinal plants were tested and some natural products
were approved as new antibacterial drug. The medicinal value of plants lies in some
chemical substances that produce a definite physiological action on the human body. The
most important of these biologically active constituents of plants are alkaloids, flavonoids,
tannins and phenolic compounds. In the last few years, a number of studies have been
conducted in different countries to prove the antimicrobial efficacy of the bioactive
compounds. However, there is still an urgent need to identify novel substances active
against pathogens with higher resistance. In this regard attempt was made to synthesize
and characterize the curcumin nanoparticles prepared using calcium carbonate treatment
of the Curcuma longa rhizome and further evaluate the antibacterial activity of the
processed rhizome extracts of Curcuma longa. In view of this fact the present study was
designed with following objectives:
OBJECTIVE:
 To study the effect of calcium carbonate treatment on recovery of curcumin.
 To characterize the formation of curcumin nanoparticles under the influence of
calcium carbonate treatment.
 To analyse the efficacy of isolated curcumin for its antimicrobial activity.

9
REVIEW OF LITERATURE: Chapter 2
Curcumin is the principal curcuminoids of the popular Indian spice turmeric, which is a
member of the ginger family (Zingiberaceae). The other two curcuminoids are
desmethoxycurcumin and bis-desmethoxycurcumin. The curcuminoids are polyphenols
and are responsible for the yellow colour of turmeric. Curcumin can exist in at least two
tautomeric forms, keto and enol. The enol form is more energetically stable in the solid
phase and in solution. Curcumin can be used for boron quantification in the so-called
curcumin method. It reacts with boric acid forming a red coloured compound, known as
rosocyanine. Curcumin is brightly yellow coloured and may be used as a food colouring.
As a food additive, its E number is E100.
Botanical Description:
Appearance- Turmeric is a perennial herbaceous plant, which reaches a stature of up to
1 meter. There are highly branched, yellow to orange, cylindrical, aromatic rhizomes . The
leaves are alternate and arranged in two rows. They are divided into leaf
sheath, petiole and leaf blade. From the leaf sheaths, a false stem is formed. The petiole is
50 to 115 cm long. The simple leaf blades are usually of a length of 76 to 115 cm and
rarely up to 230 cm. They have a width of 38 to 45 cm and are oblong to elliptic
narrowing at the tip. Inflorescence, flower and fruit- Terminally on the false stem there is a
12 to 20 cm long inflorescence stem containing many flowers. The bracts are light green
and ovate with a length of 3 to 5 centimetres to oblong with a blunt upper end. At the top
of the inflorescence stem bracts are present on which there are no flowers, these are, white
to green and sometimes tinged reddish-purple and its upper end is tapered.
The hermaphrodite flowers are zygomorphic and threefold. The three 0.8 to 1.2
centimetres long, sepals are fused, white, have fluffy hairs and the three calyx teeth are
unequal. The three bright yellow petals are fused into a corolla tube up to 3 centimetres
long. The three corolla lobes have a length of 1 to 1.5 cm, triangular with soft-spiny upper
end while the average corolla lobe is larger than the two laterals. Only the
median stamen of the inner circle is fertile. The dust bag is spurred at its base. All other
stamens are converted to staminodes. The outer staminodes are shorter than the labellum.
The labellum is yellowish, with a yellow ribbon in its centre and it is obovate, with a
length from 1.2 to 2 cm.

10
Figure 2.1: Botanical view of Curcumin longa
Biochemical Composition:
The most important chemical components of turmeric are a group of compounds
called curcuminoids, which include curcumin (diferuloylmethane), demethoxycurcumin,
and bisdemethoxycurcumin. The best studied compound is curcumin, which constitutes
3.14% (on average) of powdered turmeric. In addition there are other important volatile
oils such as turmerone, atlantone, andzingiberene. Some general constituents
are sugars, proteins, and resins.
Figure 2.2: Structure of Curcumin
The rhizome, or root, of turmeric is the part used medicinally. Numerous constituents have
been identified in turmeric. The main constituent group are polyphenolic curcuminoids
which include: curcumin (diferuloylmethane), demethoxycurcumin,
bisdemethoxycurcumin, and cyclocurcumin. The yellow-pigmented curcuminoids
represent 2% -5% of the root, typically composed of 85% as curcumin, 10% as

11
demthoxycurcumin and 5% as disdemethoxycurcumin. Curcumin is the most well studied
constituent. Turmeric also contains: sesquiterpenes (turmerone, atlantone, zingiberone,
turmeronol, germacrone, and bisabolene), carbohydrates, protein, resins and caffeic acid.
The active compound curcumin is believed to have a wide range of biological
effect including anti-inflammatory, antioxidant, anti tumour, antibacterial and antiviral
activities which indicate potential in clinical medicine.
History and traditional uses of curcumin:
The polyphenol curcumin is the active ingredient in the herbal remedy and dietary spice
turmeric (Curcu- ma longa Linn). This vibrant yellow spice, derived from the rhizome of
the plant, has a long history of use in traditional medicines of China and India. The
rhizome of turmeric has been crushed into a powder and used in Asian cookery, medicine,
cosmetics, and fabric dying for more than 2000 years. Early European explorers to the
Asian continent introduced this important spice to the Western world in the 14th century.
Use of curcumin as a folk remedy continues today. As part of the ancient Indian medical
system, Ayurveda, a poultice of turmeric paste is used to treat common eye infections, and
to dress wounds, treat bites, burns, acne and various skin diseases. The American pharma-
ceutical company Johnson & Johnson even makes turmeric Band-Aids_ for the Indian
market. In Northern India, women are given a tonic of fresh turmeric paste with powder of
dried ginger roots and honey in a glass of hot milk to drink twice daily after childbirth. A
poultice of turmeric is also applied to the perineum to aid in the healing of any lacerations
in the birth canal. Powdered turmeric is taken with boiled milk to cure cough and related
respiratory ailments, and roasted turmeric is an ingredient used as an anti dysenteric for
children. This ancient remedy is also used to treat dental diseases, digestive disorders such
as dyspepsia and acidity, indigestion, flatulence, ulcers, as well to alleviate the
hallucinatory effects of hashish and other psychotropic drugs. In food and manufacturing,
curcumin is currently used in per- fumes and as a natural yellow coloring agent, as well as
an approved food additive to flavour various types of curries and mustards.
Recent emphasis on the use of natural and complimentary medicines in Western
medicine has drawn the attention of the scientific community to this ancient remedy.
Research has revealed that curcumin has a surprisingly wide range of beneficial properties,
including anti-inflammatory, antioxidant, chemopreventive and chemotherapeutic activity.
These activities have been demonstrated both in cultured cells and in animal models, and
have paved the way for ongoing human clinical trials.

12
Properties of Curcumin: Curcumin has antioxidant, anti-inflammatory, antiviral and
antifungal actions. Studies have shown that curcumin is not toxic to humans. Curcumin
exerts anti-inflammatory activity by inhibition of a number of different molecules that
play an important role in inflammation. Turmeric is effective in reducing post-surgical
inflammation. Turmeric helps to prevent atherosclerosis by reducing the formation of
bloods clumps. Curcumin inhibits the growth of Helicobacter pylori, which causes gastric
ulcers and has been linked with gastric cancers. Curcumin can bind with heavy metals
such as cadmium and lead, thereby reducing the toxicity of these heavy metals. This
property of curcumin explains its protective action to the brain. Curcumin acts as an
inhibitor for cyclooxygenase, 5-lipoxygenase and glutathione S-transferase. It is a
common spice, known mostly for its use in Indian dishes as a common ingredient in
curries and other ethnic meals. Turmeric has also been used for centuries in Ayurvedic
medicine, which integrates the medicinal properties of herbs with food. This extraordinary
herb has found its way into the spotlight in the west because of its wide range of medicinal
benefits. Turmeric is a potent antioxidant.
Curcumin, its main active constituent, is as powerful and antioxidant as vitamins
C, E and Beta-Carotene, making turmeric usage a consumer choice for cancer prevention,
liver protection and premature aging. Several published studies also show that turmeric
inhibits the growth of several different types of cancer cells. In addition, turmeric is a
powerful anti-inflammatory, easing conditions such as bursitis, arthritis and back pain.
Turmeric’s anti-inflammatory action is likely due to a combination of three different
properties. First, turmeric lowers the production of inflammation-inducing histamine.
Secondly, it increases and prolongs the action of the body’s natural anti- inflammatory
adrenal hormone, cortisol, and finally, turmeric improves circulation, thereby flushing
toxins out of small joints where cellular wastes and inflammatory compounds are
frequently trapped. Research has also confirmed the digestive benefits of turmeric.
Turmeric acts as a cholagogue, stimulating bile production, thus, increasing the bodies’
ability to digest fats, improving digestion and eliminating toxins from the liver.
Active Constituents: The active constituents of turmeric are the flavonoid curcumin
(diferuloylmethane) and various volatile oils, including tumerone, atlantone, and
zingiberone. Other constituents include sugars, proteins, and resins. The best-researched
active constituent is curcumin, which comprises 0.3–5.4 percent of raw turmeric.
Pharmacokinetics: Pharmacokinetic studies in animals have demonstrated that 40-85
percent of an oral dose of curcumin passes through the gastrointestinal tract unchanged,

13
with most of the absorbed flavonoid being metabolized in the intestinal mucosa and liver.
Due to its low rate of absorption, curcumin is often formulated with bromelain for
increased absorption and enhanced anti- inflammatory effect.
MECHANISMS OF ACTION:
Antioxidant Effects: Water- and fat-soluble extracts of turmeric and its curcumin
component exhibit strong antioxidant activity, comparable to vitamins C and E. A study of
ischemia in the feline heart demonstrated that curcumin pretreatment decreased ischemia-
induced changes in the heart. An in vitro study measuring the effect of curcumin on
endothelial heme oxygenase-1, an inducible stress protein, was conducted utilizing bovine
aortic endothelial cells. Incubation (18 hours) with curcumin resulted in enhanced cellular
resistance to oxidative damage.
Hepatoprotective Effects: Turmeric has been found to have a hepatoprotective
characteristic similar to silymarin. Animal studies have demonstrated turmeric’s
hepatoprotective effects from a variety of he-patotoxic insults, including carbon
tetrachloride (CCl4), galactosamine, acetaminophen (paracetamol), and Aspergillus
aflatoxin. Turmeric’s hepatoprotective effect is mainly a result of its antioxidant
properties, as well as its ability to decrease the formation of pro-inflammatory cytokines.
In rats with CCl4-induced acute and subacute liver injury, curcumin administration
significantly decreased liver injury in test animals compared to controls. Turmeric extract
inhibited fungal aflatoxin production by 90 percent when given to ducklings infected with
Aspergillus parasiticus. Turmeric and curcumin also reversed biliary hyperplasia, fatty
changes, and necrosis induced by aflatoxin production. Sodium curcuminate, a salt of
curcumin, also exerts choleretic effects by increasing biliary excretion of bile salts,
cholesterol, and bilirubin, as well as increasing bile solubility, therefore possibly
preventing and treating cholelithiasis.
Anti-inflammatory Effects: The volatile oils and curcumin of Curcuma longa exhibit
potent anti-inflammatory effects. Oral administration of curcumin in instances of acute
inflammation was found to be as effective as cortisone or phenylbutazone, and one-half as
effective in cases of chronic inflammation. In rats with Freund’s adjuvant-induced
arthritis, oral administration of Curcuma longa significantly reduced inflammatory
swelling compared to controls. In monkeys, curcumin inhibited neutrophil aggregation
associated with inflammation. C. longa’s anti-inflammatory properties may be attributed
to its ability to inhibit both biosynthesis of inflammatory prostaglandins from arachidonic
acid, and neutrophil function during inflammatory states. Curcumin may also be applied

14
topically to counteract inflammation and irritation associated with inflammatory skin
conditions and allergies, although care must be used to prevent staining of clothing from
the yellow pigment.
Anticarcinogenic Effects: Animal studies involving rats and mice, as well as in vitro
studies utilizing human cell lines, have demonstrated curcumin’s ability to inhibit
carcinogenesis at three stages: tumor promotion, angiogenesis, and tumor growth. In two
studies of colon and prostate cancer, curcumin inhibited cell proliferation and tumor
growth. Turmeric and curcumin are also capable of suppressing the activity of several
common mutagens and carcinogens in a variety of cell types in both in vitro and in vivo
studies. The anticarcinogenic effects of turmeric and curcumin are due to direct
antioxidant and free-radical scavenging effects, as well as their ability to indirectly
increase glutathione levels, thereby aiding in hepatic detoxification of mutagens and
carcinogens, and inhibiting nitrosamine formation.
Antimicrobial Effects: Turmeric extract and the essential oil of Curcuma longa inhibit the
growth of a variety of bacteria, parasites, and pathogenic fungi. A study of chicks infected
with the caecal parasite Eimera maxima demonstrated that diets supplemented with 1-
percent turmeric resulted in a reduction in small intestinal lesion scores and improved
weight gain. Another animal study, in which guinea pigs were infected with either
dermatophytes, pathogenic molds, or yeast, found that topically applied turmeric oil
inhibited dermatophytes and pathogenic fungi, but neither curcumin nor turmeric oil
affected the yeast isolates. Improvements in lesions were observed in the dermatophyte-
and fungi-infected guinea pigs, and at seven days post-turmeric application the lesions
disappeared. Curcumin has also been found to have moderate activity against Plasmodium
falciparum and Leishmania major organisms.
Cardiovascular Effects: Turmeric’s protective effects on the cardiovascular system
include lowering cholesterol and triglyceride levels, decreasing susceptibility of low
density lipoprotein (LDL) to lipid peroxidation, and inhibiting platelet aggregation. These
effects have been noted even with low doses of turmeric. A study of 18 atherosclerotic
rabbits given low-dose (1.6–3.2 mg/kg body weight daily) turmeric extract demonstrated
decreased susceptibility of LDL to lipid peroxidation, in addition to lower plasma
cholesterol and triglyceride levels. The higher dose did not decrease lipid peroxidation of
LDL, but cholesterol and triglyceride level decreases were noted, although to a lesser
degree than with the lower dose. Turmeric extract’s effect on cholesterol levels may be
due to decreased cholesterol uptake in the intestines and increased conversion of

15
cholesterol to bile acids in the liver. Inhibition of platelet aggregation by C. longa
constituents is thought to be via potentiation of prostacyclin synthesis and inhibition of
thromboxane synthesis.
Gastrointestinal Effects: Constituents of Curcuma longa exert several protective effects
on the gastrointestinal tract. Sodium curcuminate inhibited intestinal spasm and p-
tolymethylcarbinol, a turmeric component, increased gastrin, secretin, bicarbonate, and
pancreatic enzyme secretion. Turmeric has also been shown to inhibit ulcer formation
caused by stress, alcohol, indomethacin, pyloric ligation, and reserpine, significantly
increasing gastric wall mucus in rats subjected to these gastrointestinal insults.
Curcumin enhances immunity: Curcumin can also help the body fight off cancer should
some cells escape apoptosis. When researchers looked at the lining of the intestine after
ingestion of curcumin, they found that CD4+ T-helper and B type immune cells were
greater in number. In addition to this localized immune stimulation, curcumin also
enhances immunity in general. Researchers in India have documented increased antibodies
and more immune action in mice given curcumin.
Curcumin blocks NF-κB and the motogenic response in Helicobacter pylori-infected
epithelial cells: Studies indicate that infection of epithelial cells by the microbial pathogen
Helicobacter pylori leads to activation of the transcription factor nuclear factor κB (NF-
κB), the induction of pro-inflammatory cytokine/chemokine genes, and the motogenic
response (cell scattering). It has been investigated that H. pylori-induced NF-κB activation
and the subsequent release of interleukin 8 (IL-8) are inhibited by curcumin
(diferuloylmethane), a yellow pigment in turmeric (Curcuma longa L.). it has been
demonstrated that curcumin inhibits IκBα degradation, the activity of IκB kinases α and β
(IKKα and β), and NF-κB DNA-binding. The mitogen-activated protein kinases (MAPK),
extracellular signal-regulated kinases 1/2 (ERK1/2) and p38, which are also activated by
H. pylori infection, are not inhibited by curcumin. It is studied that H. pylori-induced
motogenic response is blocked by curcumin. It has been concluded that curcumin, due to
inhibition of NF-κB activation and cell scattering, should be considered as a potential
therapeutic agent effective against pathogenic processes initiated by H. pylori infection.
Pregnancy and Lactation: Although there is no evidence that dietary consumption of
turmeric as a spice adversely affects pregnancy or lactation, the safety of curcumin
supplements in pregnancy and lactation has not been established.
Redox chemistry: The most common chemical studies of curcumin, aside from the
preparation of new derivatives, are those of its redox activity. The biological classification

16
of Curcumin as both pro- and antioxidant, depending on conditions, is well supported by
studies showing it to be a free radical scavenger, a reducing agent and a DNA damage
agent in the presence of Cu or Fe ions.
Nanotechnology and Curcumin: Symbiosis of ancient wisdom of the East with modern
medical science"), curcumin's unique ability to modulate multiple molecular targets makes
it a good therapeutic drug for various chronic diseases, including cancer, cardiovascular
disorders, rheumatoid arthritis, gastrointestinal disorders, neurological disorders etc. In
order to assess nano-based curcumin's potential applications in the field of medicine 254
relevant patents were analysed with segmentation based on the types of various diseases
and the data was plotted in the form of a pie chart (see figure below), which shows
disease- wise distribution of patents.
Figure 2.3: Segmentation of nano-based curcumin applications in different types of
diseases and cancers (Source: www.thomsoinnovation.com)
Curcumin shows great promise as a 'miracle drug' of the future due to its impressive array
of beneficial bioactivities. However, its therapeutic potential has not been fully exploited
because of its poor bioavailability in animals and humans. Therefore, a number of nano-
based approaches are being developed to improve curcumin's bioavailability and reduce
perceived toxicity. These approaches include solid-lipid nanoparticles, nanosuspension,
nanoemulsion, cyclodextrin curcumin self assembly, hydrogel nanoparticles, curcumin-

17
phospholipid complex and curcumin incorporated within polymer nanoparticles. The
figure below shows these various nano-based approaches for drug delivery of curcumin in
the form of a pie chart and this survey is based on 124 relevant patents for the period from
2001 to 2010. As depicted in this pie chart polymer nanoparticles play a dominant role
(34%) followed by curcumin nanoemulsion (20%), nanosuspension (13%), phospholipids
complex (12%), cyclodextrin curcumin self-assembly, hydrogel NPs and SLNs in
decreasing order. The polymer nanoparticles-incorporated drug delivery systems are
further subdivided into various classes of polymers such as generic polymers, liposomal,
PEG, micelle, PLGA, and as can be seen, generic polymers, liposomal, PEG and micelle
play a dominant role in decreasing order.

18
MATERIALS REQUIRED AND INSTRUMENTATION: Chapter 3
Materials Required:
Reagents and chemicals
Calcium Carbonate, Ethanol, Acetonitrile (HPLC grade), Orthophosphoric acid (HPLC
grade), HPLC grade Water were purchased from Merk Chemicals, Germany. Luria broth,
and Nutrient agar were purchased from Himedia, Mumbai and Curcumin (96%) from
Sigma- Aldrich, USA. All other chemicals were of reagent grade.
Herbal material
The dried Curcuma longa L., Zingiberaceae, rhizomes were purchased from the market
and were identified by Medicinal Plants Research and Development Centre (MRDC), G.B.
Pant University of Agriculture & Technology.
Other materials:
Oakridge tubes, Earthen pots, Weighing Balance, Centrifuge tubes, Grinder, Para film,
Weighing balance, Spatula, Petri plates, Spirit lamp, Spreader, Loop, Forceps, Blotting
sheets
Strains of Bacteria Used:
The different bacterial strains were collected from Department of Pathology, College of
Agriculture, G.B.P.U.A&T, Pantnagar. These strains are as follows:
 Bacillus subtilis:
Bacillus subtilis is a Gram-positive, catalase-positive bacterium. It is rod-shaped, and
has the ability to form a tough, protective endospore, allowing the organism to tolerate
extreme environmental conditions.
 Aeromonas salmonicida :
Aeromonas salmonicida is a pathogenic bacterium that severely impacts salmonid
populations and other species. Aeromonas salmonicida’s ability to infect a variety of
host, multiply, and adapt, make it a prime virulent bacterium. A. salmonicida is an
etiological agent for furunculosis; a disease that causes septicemia, haemorrhages,
muscle lesions, inflammation of the lower intestine, spleen enlargement, and death in
freshwater fish populations.
 Aeromonas hydrophila :

19
Aeromonas hydrophila is a heterotrophic, Gram-negative, rod-shaped bacterium mainly
found in areas with a warm climate. This bacterium can be found in fresh or brackish
water. It can survive in aerobic and anaerobic environments, and can digest materials
such as gelatin and haemoglobin.
 Escherichia coli :
Escherichia coli is a gram-negative, facultatively anaerobic, rod-shaped bacterium of
the genus Escherichia that is commonly found in the lower intestine of warm-
blooded organisms (endotherms).
Figure 3.1: Bacterial Cell Suspension Cultures
Instrumentation used during Research:
 Zeta Sizer:
The Zetasizer Nano ZS90 is the perfect lower cost solution when the ultimate in sizing
sensitivity is not necessary, or where identical results to a legacy system with 90 degree
scattering optics is required. It is an entry level system for the measurement of particle
size and molecular size at a 90 degree scattering angle using Dynamic Light Scattering,
also with the ability to measure zeta potential and electrophoretic mobility using Laser
Doppler Microelectrophoresis, and molecular weight using Static Light Scattering.
It is entirely based on the principle of dynamic scattering of light.
Dynamic light scattering (DLS), sometimes referred to as Quasi-Elastic Light
Scattering (QELS), is a non-invasive, well-established technique for measuring the
size and size distribution of molecules and particles typically in the submicron
region, and with the latest technology lower than 1nm.Typical applications of
dynamic light scattering are the characterization of particles, emulsions or

20
molecules, which have been dispersed or dissolved in a liquid. The Brownian
motion of particles or molecules in suspension causes laser light to be scattered at
different intensities. Analysis of these intensity fluctuations yields the velocity of
the Brownian motion and hence the particle size using the Stokes-Einstein
relationship.
Dynamic light scattering technology offers the following advantages:
 Accurate, reliable and repeatable particle size analysis in one or two minutes.
 Measurement in the native environment of the material.
 Mean size only requires knowledge of the viscosity of the liquid.
 Simple or no sample preparation, high concentration, turbid samples can be
measured directly.
 Simple set up and fully automated measurement.
 Size measurement of sizes < 1nm.
 Size measurement of molecules with MW < 1000Da.
 Low volume requirement (as little as 2µL).
Figure 3.2: Zeta Sizer

21
 Ultrasonicator:
Sonication is the act of applying sound energy to agitate particles in a sample, for
various purposes. Ultrasonic frequencies (>20 kHz) are usually used, leading to the
process also being known as ultrasonication or ultra-sonication. In the laboratory, it is
usually applied using an ultrasonic bath or an ultrasonic probe, colloquially known as
a sonicator. In a paper machine, an ultrasonic foil can distribute cellulose fibres more
uniformly and strengthen the paper.
Sonication has numerous effects, both chemical and physical. The chemical effects
of ultrasound are concerned with understanding the effect of sonic waves on chemical
systems. The chemical effects of ultrasound do not come from a direct interaction with
molecular species. In biological applications, sonication may be sufficient to disrupt or
deactivate a biological material. For example, Sonication is often used to disrupt cell
membranes and release cellular contents. Sonication is also used to fragment molecules
of DNA, in which the DNA subjected to brief periods of sonication is sheared into
smaller fragments.
 Magnetic stirrer:
A magnetic stirrer or magnetic mixer is a laboratory device that employs a rotating
magnetic field to cause a stir bar Since glass does not affect a magnetic
field appreciably (it is transparent to magnetism), and most chemical reactions take
place in glass vessels (i.e. see beaker (glassware) or laboratory flasks), magnetic stir
bars work well in glass vessels immersed in a liquid to spin very quickly, thus stirring
it. The rotating field may be created either by a rotating magnet or a set of stationary
electromagnets, placed beneath the vessel with the liquid.
 High Performance Liquid Chromatography:
High-performance liquid chromatography (sometimes referred to as high-pressure
liquid chromatography), HPLC (or just LC), is a chromatographic technique used to
separate a mixture of compounds in analytical chemistry and biochemistry with the
purpose of identifying, quantifying or purifying the individual components of the
mixture. HPLC is considered an instrumental technique of analytical chemistry (as
opposed to a gravitimetric technique). HPLC has many uses including medical (e.g.
detecting vitamin D levels in blood serum), legal (e.g. detecting performance

22
enhancement drugs in urine), research (e.g. separating the components of a complex
biological sample, or of similar synthetic chemicals from each other), and
manufacturing (e.g. during the production process of pharmaceutical and biological
products).
Chromatography can be described as a mass transfer process involving adsorption.
HPLC relies on pumps to pass a pressurized liquid and a sample mixture through a
column filled with a sorbent, leading to the separation of the sample components. The
active component of the column, the sorbent, is typically a granular material made of
solid particles (e.g. silica, polymers, etc.), 2-50 micrometers in size. The components of
the sample mixture are separated from each other due to their different degrees of
interaction with the sorbent particles. The pressurized liquid is typically a mixture of
solvents (e.g. orthophosphoric acid, acetonitrile) and is referred to as "mobile phase".
Its composition and temperature plays a major role in the separation process by
influencing the interactions taking place between sample components and sorbent.
These interactions are physical in nature, such as hydrophobic (dispersive), dipole-
dipole and ionic, most often a combination there of.
The schematic of an HPLC instrument typically includes a sampler, pumps, and a
detector. The sampler brings the sample mixture into the mobile phase stream which
carries it into the column. The pumps deliver the desired flow and composition of the
mobile phase through the column. The detector generates a signal proportional to the
amount of sample component emerging from the column, hence allowing for
quantitative analysis of the sample components. A digital microprocessor and user
software control the HPLC instrument and provide data analysis. Some models of
mechanical pumps in a HPLC instrument can mix multiple solvents together in ratios
changing in time, generating a composition gradient in the mobile phase. Various
detectors are in common use, such as UV/Vis, photodiode array (PDA) or based on
mass spectrometry. Most HPLC instruments also have a column oven that allows for
adjusting the temperature the separation is performed at.

23
Figure 3.3: HPLC
 Scanning Electron Microscope (SEM)
A Scanning Electron Microscope (SEM) is a type of electron microscope that produces
images of a sample by scanning it with a focused beam of electrons. The electrons interact
with atoms in the sample, producing various signals that can be detected and that contain
information about the sample's surface topography and composition. The electron beam is
generally scanned in a raster scan pattern, and the beam's position is combined with the
detected signal to produce an image. SEM can achieve resolution better than 1 nanometer.
Specimens can be observed in high vacuum, in low vacuum, (in environmental SEM) in
wet conditions and at a wide range of cryogenic or elevated temperatures.
The most common mode of detection is by secondary electrons emitted by atoms
excited by the electron beam. The number of secondary electrons is a function of the angle
between the surface and the beam. On a flat surface, the plume of secondary electrons is
mostly contained by the sample, but on a tilted surface, the plume is partially exposed and
more electrons are emitted. By scanning the sample and detecting the secondary electrons,
an image displaying the tilt of the surface is created.

24
Figure 3.4: Scanning Electron Microscope
METHODOLOGY:
A.SAMPLE PREPARATION OF Curcuma longa:
 Protocol:
a) Six earthen pots with lids were taken and washed with autoclaved distilled water.
b) Six samples each of 10 gm were weighed for sample preparation.
c) The earthen pots were marked as following:
SAMPLES
1 day control
3 day control
7 day control
1 day sample
3 day sample
7 day sample

25
d) Then equivalently weighed Curcuma longa extract powder was dissolved in case of 1,
3 and 7 days control in autoclaved distilled water.
e) While in case of samples, equivalent amount of calcium carbonate was weighed for the
1, 3 and 7 days sample in respect to weighed Curcuma longa extract powder.
f) They were stored in dark place.
g) Water level is daily checked and maintained.
h) After 1 day completion, the sample was removed from pot of 1 day control and 1 day
sample.
i) Then it was washed thrice with autoclaved distilled water.
j) The sample was then dried and crushed in grinder.
k) The tubes were labelled and parafilmmed then.
l) Similar is the procedure that was carried for 3 and 7 days sample.
B. SYNTHESIS AND CHARACTERIZATION OF CURCUMIN NANO
PARTICLES THROUGH ZETA SIZER:
 Protocol:
1. Preparation of sample:
 The crushed samples (100 mg) of Curcuma longa extract were taken separately along
with the control and calcium carbonate treated Curcuma longa extract.
 25 ml of autoclaved distilled water was added in a conical flask.
 After that it was kept on magnetic stirrer for 3 hours at room temperature, followed by
half an hour at 50ºC.
 After cooling the samples, the samples were transferred in centrifuge tubes.
 Let it spin down at 4ºC at 10000 rpm for 10 minutes.
 Supernatant was taken in a separate tube and then stored at 4ºC.
 Place them in ultrasonicator at 30ºC for 5 minutes in floater.
2. Loading in ZETA SIZER:
 The cuvette was then rinsed with distilled water.
 The standard was set with autoclaved distilled water.
 The samples were loaded in cuvette one by one for analysis through Zeta Sizer.
C. ASSESSMENT OF ANTI MICROBIAL PROPERTY OF Curcuma longa:
 Protocol:
 20 µl of each day of prepared sample was taken as described above.

26
 20 µl of water to be used as a second control was taken.
Reviving of bacterial suspension culture:
 Suspension solutions of 25 ml each for 4 bacterial samples were made from Luria
Broth.
 The suspension solutions were then autoclaved.
 The bacteria from the plate was taken with the help of a loop and inoculated then in
the suspension solutions separately for four samples under laminar flow.
 After that they were kept at 37ºC for 16 hours in shaker.
 Nutrient agar plates (approx 25 of 20 ml each) that are to be used for spreading
and streaking were made.
 The plates were checked for contamination by placing them under laminar flow for 3-4
hours.
 As a precautionary measure for contamination, the plates were made one day prior to
inoculation.
 Then under laminar flow, 100µl of the bacterial suspension was spread on the nutrient
agar plate with the help of spreader separately for 4 bacterial samples.
 For streaking, a loop from bacterial suspension was taken and then streaked on nutrient
agar plate separately for 4 bacterial samples.
 They were then placed at 37ºC for 16 hours in incubator.
 The spreading experiment was repeated until the uniform growth was observed.
1. Plating:
 Laminar flow was sterilized by opening UV light fifteen minutes prior to conducting
of experiment.
 A nutrient agar contamination free plate was taken and then spreading was done with
16 hours earlier bacterial suspension culture.
 The discs of blotting sheet were autoclaved. It was used as a medium to keep the
turmeric sample. Although disc method can also be used.
 Then four quadrants were made on Petri plate and then labelled as water, methanol, 7
day control and 7 day Calcium Carbonate treated sample.
 The discs were then saturated by 10 µl first followed by another 10 µl.
 The discs were placed with the help of forceps on the quadrants made at the Petri plate.
 The plates were parafilmmed and then kept at 37ºC for overnight in incubator.
 The growth was then observed after 16 hours and minimum inhibitory concentration
(MIC) was determined.

27
D. QUANTIFICATION USING HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY:
 Protocol:
 Curcumin analytical standard required to make 1000 ppm solution was weighed.
 After that solutions f 20µl, 40µl, 60µl, 80µl and 100µl were made from that 1000 ppm
one.
 2 mg of control and calcium carbonate treated turmeric samples were weighed (1, 3
and 7 days) separately.
 After that they were dissolved in centrifuge tube in 2µl of methanol each.
 Then they were kept for vortexing for 30 minutes. The colour was observed.
 Centrifugation at 10,000 rpm for 5 minutes was done.
 Followed by filtration with 0.45µm filter using a syringe.
 Then they were placed in different centrifuge tube.
2. Preparation of mobile phase and stationary phase:
The phase consisted of acetonitrile and 0.1% orthophosphoric acid in the ratio of 60:40
v/v. Both the solvents were filtered using 0.45µm filter paper. Then the degassing of the
solvent was done in ultrasonicator at 25ºC for 15 minutes. The isocratic elution was
carried out at flow rate of 1 ml/min at ambient temperature and at a wavelength of 425 nm.
3. Loading of HPLC:
 The system was pre run with mobile phase that consisted of acetonitrile and 0.1%
orthophosphoric acid in the ratio of 60:40 v/v.
 Run for 1 hour with acetonitrile and 0.1% OPA was given.
 The samples of curcumin analytical standard were loaded twice each so as to obtain a
sharp peak and then the standard was set for knowing the unknown concentration.
 Calibration of the obtained curve was done.
 After this, the control and calcium carbonate treated samples were loaded, one by one,
each three times.
 The peaks were then observed.
 Calibration and analysis of the result was done.

28
RESULT AND DISCUSSION: Chapter 4
The turmeric sample was prepared as described in methodology sections. It was observed
that the samples of turmeric after crushing had the colour difference according to the
increment in the number of days for reaction with calcium carbonate. A clear cut colour
difference was observed (Figure 4. 1). They were stored in centrifuge tubes after putting
parafilm for further characterization.
Figure 4.1: Powdered form of Curcuma longa extract after treatment with calcium
carbonate at 1 day, 3 days and 7 days of treatment
2. Estimation of curcumin content using high performance liquid chromatography:
Processed extract of Curcuma longa was analysed with HPLC. The turmeric sample was
prepared as described in methodology sections. HPLC analysis was performed comprising
a pump, a manual sampler and a photodiode array (PDA) detector. Chromatographic
separation was carried out with a silica column. The mobile phase, which composed of
acetonitrile and 0.1% orthophosphoric acid in ratio of 60: 40 and adjusted to 2.7 pH and
was set at an isocratic mode with a flow rate of 1.0 mL/min. The detection wavelength
was 425 nm. The injection volume was 50.0 μl and the total run time was fixed at 15 min.
Data acquisition and analysis were performed by using EZCHROME elite
software.

29
The calibration curve was constructed by the dilution of curcumin standard
(Sigma-Aldrich , Germany) with methanol to provide the desired concentrations
(20ppm,40ppm,60ppm,80ppm,100ppm) followed by injection into the HPLC system.
Samples were directly dissolved in HPLC grade methanol/water and prior to injection in
the LC system, both standard solutions and samples were filtered through 0.45 μm
(Millipore, Brazil) membranes. The curcumin content in dry basis was calculated based on
dried mass of extractives for each extract. The soluble solids yield and curcumin yield
were calculated according.
As the maximum solubility of curcumin occurs in methanol therefore samples
were prepared in HPLC ultra pure grade methanol solvent. Moreover, the solubility also
decreases with the increment in number of days for treatment with calcium carbonate.It
was observed that the colour depicted in control is consistent even as the numbers of days
are increased. While in the case of calcium carbonate treated samples, more the treatment
with the calcium carbonate less is the solubility. There is a significant colour difference
that can be observed in 1 day control and treated sample, 3 day control and treated sample
and 7 day control and treated sample respectively. With this analysis, one can infer that
there is capping in case of calcium carbonate treated samples due to which the solubility
factor is decreasing. On loading the curcumin analytical standard at different
concentrations of 20ppm, 40ppm, 60ppm, 80ppm, 100 ppm, 200 ppm and 400 ppm; we
get the standard curve as shown in Figure 4.2.

30
Figure 4.2: Standard analytical Curcumin curve
After running the unknown calcium carbonate treated samples and control, we get
the plot area. Therefore with the use of equation determined from standard curve, we get the
concentration of curcumin as shown in table 4.1.
Table 4.1: Quantitative estimation of curcumin content in extract of Curcuma longa after
treatment with calcium carbonate for three different time intervals (1, 3 and 7 days of
treatment)
Samples Peak area (y%106
) Concentration(x)
in mg/ml
1 DT 9.145230 0.003370
3 DT 2.096167 0.000772
7 DT 0.585204 0.000215
1 DC 22.34480 0.008236
3 DC 18.98830 0.006999
7 DC 12.56367 0.004630
Where DT corresponds to Day Treatment
DC corresponds to Day Control

31
Figure 4.3: HPLC Analysis of the 1 DC of Curcuma longa rhizome.
Figure 4.4: HPLC Analysis of the 1 DT Curcuma longa rhizome treated with calcium carbonate

32
Figure 4.5: HPLC Analysis of the 3 DC of Curcuma longa rhizome

33
Figure 4.7: HPLC Analysis of the 7 DC of Curcuma longa rhizome

34
Moreover, the control and samples treated with calcium carbonate were also dissolved in
HPLC grade water to determine the solubility of curcumin in water in the processed extract as
well as control sample. The results obtained are as follows:
Samples Peak area(y%106
) Concentration(x)
In mg/ml
1 DC at 10000rpm 16.371734 0.006034
1 DT at 10000rpm 0.722199 0.000266
Figure 4.9: HPLC Analysis of the 1 DC Curcuma longa rhizome after centrifugation at 10000 rpm

35
after centrifugation at 10000 rpm
3. Synthesis and characterisation of nanoparticles using Calcium Carbonate:
Particle size and size distribution of synthesized nanoparticle from processed extract of
curcuma longa were determined by using Zetasizer. The control and calcium carbonate treated
samples of curcuma longa were dissolved in autoclaved distilled water and prepared as
described in methodology section. Interestingly, through Dynamic Light Scattering it was
observed that single peak was formed in control sample of Curcuma longa rhizome extract.
However, two sized nanoparticles were observed in samples of curcuma longa rhizome extract
which were treated with calcium carbonate. The average particle size of 1, 3, 7 days control
sample were 138nm, 158nm, 138nm respectively and in 1,3,7 days treated samples two size
of particle (227nm,78nm), (278nm,51nm),(224,51nm) respectively are observed. The particle
size in case of treated samples indicated stability. While the intensity of peak size obtained
from nanoparticle formed from extracted sample of Curcuma longa was reduced as compared
to nanoparticle formed from control sample of Curcuma longa. It is observed that there is
formation of smaller nanoparticles (50-80 nm) in treated sample which was also having a
bigger particle (220-280 nm). The size distribution curve indicates that the particle is getting
inter converted into the smaller particles.

36
The increase in size points towards either capping of curcumin particle due to treatment
of calcium carbonate which is also resulting in its stability or in the formation of big sized
particle and another small sized particle as seen in the size distribution curve for 1, 3 and 7
days calcium carbonate treated samples (Figure 4.12.). It further points towards the inter
conversion of the bigger particles into the smaller ones due to the derivatisation of curcumin by
calcium carbonate treatment. By analysis of the result obtained, we also infer that there is
decrease in size of curcumin nanoparticles after treatment with calcium carbonate.
1 day control (138nm) 3 day control (158nm)
7 day control (138nm)
Figure 4.11: Size distribution curve of 1, 3 and 7 day control of Curcuma longa rhizome

37
1 day sample (227nm) 3 day sample (278nm)
7 day sample (224nm)
Figure 4.12: Size distribution curve 1, 3, 7 day samples of Curcuma longa rhizome treated
with calcium carbonate
4. Analysis of the synthesized nanoparticles prepared from curcuma longa rhizome
treated with Calcium Carbonate using Scanning Electron Microscopy
Scanning Electron Microscopy was performed to analyse the surface and size of
nanoparticles found from the curcumin. The control and calcium carbonate treated samples of
curcumin longa extract were dissolved in autoclaved distilled water and prepared as
described in methodology section and subjected for Scanning Electron Microscopy. One day
control showing the homogenous nanoparticle of average size 138nm and one day sample
showing the heterogenous nanoparticle which size ranging from 80nm-200nm. The result
suggested that there is reduction in size of nanoparticles occurs after treatment with calcium
carbonate. Further experiment needed to assess the formation of nanoparticles with long time
interval of exposure with calcium carbonate.

38
Figure 4.13: SEM image for 1 DC sample extract from curcuma longa rhizome at 6500x
Figure 4.14: SEM image for 1 DT sample extract from curcuma longa rhizome which is
treated with Calcium Carbonate at 6500x

39
5. Assessment of Antimicrobial Activity depicted by turmeric samples:
Curcuma longa rhizome has been traditionally used as antimicrobial agent as well as an
insect repellent. Several studies have reported the broad-spectrum antimicrobial activity
for curcumin including antibacterial, antiviral, antifungal, and antimalarial activities.
Because of the extended antimicrobial activity of curcumin and safety property even at
high doses (12 g/day) assessed by clinical trials in human, it was used as a structural
sample to design the new antimicrobial agents with modified and increased antimicrobial
activities through the synthesis of various derivatives related to curcumin. In the present
study we assessed the comparative anti-bacterial properties of extracts processed extract of
curcuma longa on four different bacterial strains
The four bacterial strains were taken to check the growth inhibition activity of the
curcumin as prepared. Both the control and calcium carbonate treated samples were used.
A clear cut boundary inhibition was seen as there was no growth in that periphery. Three
out of four strains could show the differentiated boundary near the sample saturated disc.
We can infer the boundary dimension as created by the samples presence around the disc.
A significant growth inhibition could be seen and shown in (figure: 4.13)
. According to the analysis, curcumin samples showed maximum growth inhibition
in E. coli 40 followed by in Aeromonas Hydrophilla and then A. Salmonicida and B.
Subtilis.
Figure 4.15: Boundary around the saturated disc depicting inhibition of growth

40
Figure 4.16: Anti-bacterial study: inhibition in growth in four bacterial strains challenged
with curcumin extract as control and processed curcuma longa extract sample

41
SUMMARY CONCLUSION: Chapter 4
With the aim of unwinding the hidden characteristics of turmeric, we performed the
experiments of synthesis, characterization and anti microbial assay for nanoparticles prepared
from curcuma longa rhizome. The results obtained provided new trends that can be further
confirmed by performing better insights for preparation and their applications. The results
obtained are as below:
 There was a colour difference obtained in the control and calcium carbonate
treated rhizomes of Curcuma longa with respect to 1, 3 and 7 days of treatment.
 The HPLC analysis showed that the concentration of curcumin content decreased
in the calcium carbonate treated rhizomes as the number of days were increased for
treatment with calcium carbonate.
 The methanol solubility of the Curcumin in treated sample decreased with the
increase in number of the days given for the calcium carbonate treatment. The
main possible reason behind the decrease in solubility can be credited to the
capping of the curcumin particles or formation of the aggregates. Further, it may be
due to the formation of smaller sized nanoparticles encapsulating curcumin which
are not solubilised in methanol.
 As far the Zeta Sizer experiment is concerned, the analysis concluded that while
comparing size distribution curve for 1, 3 and 7 days extract of Curcuma longa
rhizome which was treated with calcium carbonate, we find the formation of a two
sized nanoparticle. The size distribution curve indicates towards the inter
conversion of the aggregates into the derivatising forms of curcumin. This
formation needs further experimentation like FITR so as to lead to a final
conclusion of what it is. However it is clearly observed that size of nanoparticle
reduces with increase in treatment of calcium carbonate.
 Control and calcium carbonate treated samples depicted inhibition of growth for
the strains A. salmonicida, B. subtilis, E. coli 40 and Aeromonas hydrophilla. The
inhibition was significantly higher in treated samples as compared to control
The interesting results were obtained in preliminary attempts, however to understand the
mechanism behind the reduction in size of nano-particles formed from Curcuma longa
extract treated with calcium carbonate needs further study to be done.

42
REFERENCES:
1. Bush J.A., Cheung K.J. Jr, Li G., 2001, Curcumin induces apoptosis in human
melanoma cells through a Fas recep-tor/caspase-8 pathway independent of p53. Exp Cell
Res, 271: 305-314.
2. Cheng A.L., Hsu C.H., Lin J.K., 2001, Phase I clinical trial of curcumin, a
chemopreventive agent, in patients with high-risk or pre-malignant lesions. Anticancer
Res, 21: 2895-2900.
3.Hanif R., Qiao L., Shiff S.J., Rigas B., 1997, Curcumin, a natural plant phenolic food
additive, inhibits cell proliferation and induces cell cycle changes in colon
adenocarcinoma cell lines by a prostaglandin-independent pathway. J Lab Clin Med, 130:
576-584.
4. Hour T.C., Chen J., Huang C.Y., 2002, Curcumin enhances cytotoxicity of
chemotherapeutic agents in pros-tate cancer cells by inducing p21 (WAF1/CIP1) and
C/EBPbeta expressions and suppressing NF-kappa activation. Prostate, 51: 211-218.
5. Kawamori T., Lubet R., Steele V.E., 1999, Chemopreventative effect of curcumin, a
naturally occurring anti-inflammatory agent, during the promotion/progression stages of
colon cancer. Cancer Res, 59: 597-601.
6. Lal B., Kapoor A.K., Asthana O.P., 1999, Efficacy of curcumin in the management of
chronic anterior uveitis. Phytother Res, 13: 318-322.
7. Limtrakul P., Lipigorngoson S., Namwong O., 1997, Inhibitory effect of dietary
curcumin on skin carcino-genesis in mice. Cancer Lett, 116: 197-203.
8. Mehta R.G., Moon R.C., 1991, Characterization of effective chemopreventive agents in
mammary gland in vitro using an initiation-promotion protocol. Anticancer Res, 11: 593-
596.
9. Mukhopadhyay A., Basu N., Ghatak N., 1982, Anti-inflammatory and irritant activities
of curcumin analogues in rats. Agents Actions, 12: 508-515.
10 .Munzenmaier A., et al., 1997, A secreted/shed product of Helicobacter pylori activates
transcription factor nuclear factor-kappa B. J Immunol, 159: 6140-47.
11. Park EJ, et al., 2000, Protective effect of curcumin in rat liver injury induced by carbon
tetrachloride. J Pharm Pharmacol, 52: 437-40.
12. Pendurthi U.R., et al., 1997, Inhibition of tissue factor gene activation in cultured
endothelial cells by curcumin. Suppression of activation of transcription factors Egr-1,
AP-1, and NF-kappa B. Arterioscler Thromb Vasc Biol., 17: 3406-13.
13.Ramachandran C., Fonseca H.B., Jhabvala P., et al., 2002, Curcumin inhibits
telomerase activity through human telomerase reverse transcriptase in MCF-7 breast

43
cancer cell line. Cancer Lett, 184: 1-6.
14. Reddy B.S., Rao C.V., 2002, Novel approaches for colon cancer prevention by
cyclooxygenase- 2 inhibitors. J Environ Pathol Toxicol Oncol, 21: 155-164.
15. Shao Z.M., Shen Z.Z., Liu C.H., et al., 2002, Curcumin exerts multiple suppressive
effects on human breast carcinoma cells., Int J Cancer, 98: 234-240.
16. Sharma A., et al., 2000, Spice extracts as dose-modifying factors in radiation
inactivation of bacteria. J Agric Food Chem, 48: 1340-44.
17. Simon A, et al., 1998. Inhibitory effect of curcuminoids on MCF-7 cell proliferation
and structure-activity relationships. Cancer Lett, 129:111-16.
18. Somasundaram S., Edmund N.A., Moore D.T., et al., 2002, Dietary curcumin inhibits
chemotherapy-induced apop-tosis in models of human breast cancer. Cancer Res, 62:
3868- 3875.
19. Sreejayan, N. & Rao, M.N., 1994, Curcuminoids as potent inhibitors of lipid
peroxidation. J. Pharm.Pharmacol, 46: 1013.
20. Sreejayan, N., & Rao, M.N. 1996, Free radical scavenging activity of curcuminoids.
21. Srivastava R., Puri V., Srimal R.C., Dhawan B.N. 1986, Effect of curcumin on platelet
aggregation and vascular prostacyclin synthesis. Arzneimittelforschung, 36: 715-717.
22. Thaloor D., Singh A.K., Sidhu G.S., et al., 1998, Inhibition of angiogenic
differentiation of human umbilical vein endothelial cells by curcumin. Cell Growth Differ,
9: 305-312.
23. Venkatesan N., 2000, Pulmonary protective effects of curcumin against paraquat
toxicity. Life Sci 66(2):PL21-28.
24 .Verma S.P., et al., 1997, Curcumin and genistein, plant natural products, show
synergistic inhibitory effects on the growth of human breast cancer MCF-7 cells induced
by estrogenic pesticides. Biochem Biophy Res Comm, 233: 692-96.
25.
Verma S.P., et al., 1998, The inhibition of the estrogenic effects of pesticides and
environmental chemicals by curcumin and isoflavonoids. Environ Health Perpect, 106:
807-812.

B.Tech Thesis

Recommandé

Recommandé

Contenu connexe

Tendances

Tendances (20)

Similaire à B.Tech Thesis

Similaire à B.Tech Thesis (20)

B.Tech Thesis