Thesis

1. MICROWAVE ASSISTED GREEN SYNTHESIS OF ALUMINIUM NANOPARTICLES
USING LEAF EXTRACT OF Cinnamomum tamala AND Solanum nigrum FOR
ANTIMICROBIAL ACTIVITY
A Thesis Submitted
In Partial Fulfillment of the Requirements
for the Degree of
MASTER OF PHARMACY
in
Pharmaceutics
by
ASHISH KUMAR GUPTA
(Enrollment No.1412350028725)
Under the Supervision of
A Thesis Submitted In Partial Fulfillment of the Requirements For the Degree of
MASTER OF PHARMACY
in
PHARMACEUTICS
by
ASHISH KUMAR GUPTA
(Enrollment No. 1412350028725)
Under the supervision of
Dr. Shekhar Singh Mrs. Geeta Mishra
Assoc. Professor (Pharmacy) Assoc. Professor (Pharmacy)
B.B.D.N.I.I.T, LUCKNOW B.B.D.N.I.I.T, LUCKNOW
To the
DR. APJ ABDUL KALAM TECHNICAL UNIVERSITY
LUCKNOW
(Formerly Uttar Pradesh Technical University, Lucknow)
May, 2021
1

2. Introduction
Microbes are another name of microorganisms. Microorganisms such as bacteria, fungi, viruses, etc.
which are found as single-cell or cell colony. These tiny insects are invisible to naked eye but can see
under a microscope.
The cell life form was divided into 3 sub-systems, namely archaea, bacteria (prokaryote) and
eukaryote.
• Archaea (Prokaryotic): they do not have nuclear membrane. Such as: methanogen (produce
methane gas).
• Bacteria (Prokaryotic): These organisms do not have nuclear membrane. Mainly diacyl glycerol
diester lipid are present in their membrane. Such as: Cyanobacteria (photosynthesizing bacteria),
spirochetes (Gram-negative).
• Eukaryote: They are multicellular organism and have nuclear. Its further divides into three
categories:
Kingdom Animalia- Example: Arthropoda (it contains insects, arachnid and crustacean), chordate
(vertebrates).
Kingdom Fungi – Example: Saccharomyces (true yeast), Basidiomycota includes mushrooms.
Kingdom Plantae- Example: Bryophyta (mosses), Magnoliophyte (flowering plant). 2

3. Antimicrobial agents
 The antibacterial and antifungal properties of aluminum nanoparticles are well known in
agriculture and the pharmaceutical industry. The combination of Salvarsan (the first antimicrobial
agent) was discovery of penicillin in 1928. Penicillin is a fungal metabolite, used as an antibiotic.
Antimicrobial is definite as "any herbal, synthetic & semi-synthetic origin, which kills or stops the
growth of micro-organism during low concentration, but causes slightly or no institutional
damage’’. Bacteriostatic and bactericidal terms to indicate anti-bacterial activity.
 MIC is the lowermost concentration of anti-microbial agent requested to inhibit pathogen growth,
in other words, bacteriostatic are the main agents responsible for stopping bacterial growing by
keeping them in a stable growth stage. Bactericidal means, kills germs. Anti-bacterial agents are
available in markets with potentially bactericidal & bacteriostatic properties.
 In current years, many iron ions have studied as antibacterial agents in research. These metal ions
include aluminum, silver, copper, zinc, ferric, magnesium and titanium.
3

4. LITERATURE REVIEW
• Nigussie et al (2021) antimicrobial activity of Lawsonia inermis, Azadirachta indica and Aryranthes
aspera extract against Staphylococcus aureus, Streptococcus pyogenes, E-coli, Klebsiella
pneumoniae, Pseudomonas aeruginosa and Shawnelle algae. The result was showed extracts of
Azadirachta indica was showed better antimicrobial activity against all bacterial species as
compared to Lawsonia inermis and Aryranthes aspera extract.
• Altiuwiriqi et al (2020) synthesized aluminum nanoparticles by using Pulsed laser ablation
techniques. Prepared nanoparticles were characterized by TEM, particles size, zeta potential and
PDI. The result was showed good particle size and crystalline structure. It was concluded that the
Vinegar Al- NPs slight existence of Al2O3 NPs in the Suspension.
• Prepared nanoparticle of Aluminum and Nickel oxide by Chahardoli et al (2020) for antioxidant
activity. The prepared nanoparticles were characterized by particle size, XRD, UV-Visible
spectroscopy. It was concluded that the synthesized aluminum nanoparticles having significant
potential for antioxidant activity compared to nickel oxide nanoparticles.
4

5. • Prepared silver nanoparticles using extracts of Aegle marmelos by Devi et al (2020) for the
antimicrobial activity against B. cereus and S. dysentrial. The prepared nanoparticles were
characterized by particle size, XRD, UV-Visible spectroscopy and results showed an intense SPR
band was 159 and 181 nm. The study concluded that the silver nanoparticles were shown to be
effective against B. cereus and S. dysentrial.
• Mohammed et al (2020) formulated topical gel with different polymers (HPMC, Carbopol &
Sodium alginate) for antibacterial activity. The prepared gel was evaluated by viscosity, pH, In-vitro
release. The results showed good viscosity and better antimicrobial activity.
• Silver nanoparticles using the aqueous extract of leaves of Cinnamomum tamala were synthesized by
Nahar et al (2020) for antibacterial activity against Bacillus subtilis and Staphylococcus aureus. The
prepared AgNPS were observed by various parameters. UV-Visible spectroscopy scanning, the SPR
was observed at 9-16 nm and crystalline shape. Cinnamomum tamala loaded silver nanoparticles
exhibited effective antibacterial activity against Bacillus subtilis and Staphylococcus aureus.
• Das et al (2020) Prepared a Cinnamomum tamala leaves extract loaded magnetite nanoparticles for
the antibacterial activity against E- coli and S. aureus. The prepared nanoparticles were characterized
by particle size, zeta potential, FTIR, XRD and SEM. The result was revealed 26-42 nm particles size
was found to be size and also showed better antibacterial activity.
5

6. RESEARCH ENVISAGED
Microbial infection are serious health problems, it causing many types of dead diseases in human and
animals. At a present time, many synthetic chemicals containing products are available and frequently
used as antimicrobial agents. Due to using synthetic products different types of adverse effects have
been reported like sensory disturbances, skin patches, skin swelling and skin allergies. To overcome
the limitation of Synthestic chemicals, nowadays plants derived formulation and natural oils are as an
alternative to microbial control due to it safer and cost effective. Medicinal plants Cinnamomum
tamala and Solanum nigrum have alkaloid, flavonoids, steroids, terpenoids shows on effective against
antimicrobial activity.
Cinnamomum tamala and Solanum nigrum extracts is an effective therapy in the treatment of
microbial infection at pathological sites offer great advantage in fast release of drug and directly to site
of action.
This research work is design to Synthesis of Aluminium nanoparticles of plant extracts for
antimicrobial activity. Aluminium also has antimicrobial properties.
The aim of the study is to developed a Cinnamomum tamala and Solanum nigrum extracts loaded
Aluminium nanoparticles to treat antimicrobial activity.
6

7. Plant Profile
(a) Solanum nigrum (Makoi)
Taxonomical Classification
Kingdam -Plantae
Order - Solanales
Family – Solanaceae
Genus – Solanum
Species – Solanum nigrum
Chemical constituents
Steroidal glycosides, Steroidal alkaloids, Steroidal oligoglycosides, including
solamargine, solasonine, solavilline, solasdamine, and solanine, steroidal saponins and glycoprotein,
many polyphenolic compounds such as gallic acid, protocatechuic acid, catechin, caffeic acid,
epicatechin, rutin, and naringenin.
Medicinal use
antioxidant, antimicrobial, antivirus, anticancer, antitumor, hepato-protective, cardio-
protective, cytotoxic activity, anti inflammatory, anti-seizure activity and larvicidal effects.
7

8. (b) Cinnamomum tamala (Tej Patta)
Taxonomical Classification
Kingdom – Plantae
Order – Laurales
Family - Lauraceae
Genus - Cinnamomum
Species – Cinnamomum tamala
Chemical Constituents
α-pinene, camphene, myrcene, limonene, eugenol, p-cymene, methyl eugenol, eugenol
acetate, methyl ether of eugenol, beta-caryophyllene, linalool and caryophyllene oxide.
Medicinal use
antimicrobial, dyspeptic complaints, cough and bronchitis, inflammation of the mouth
and pharynx, antidiabetic,, loss of appetite, antioxidant, fever and cold, antiulcer and also used in
antimicrobial activity and larvicidal effect.
8

9. PLAN OF WORK
Pre-formulation
1.Collection of Plant Material.
2.Identification of the Plant Material.
3.Preparation of Extract.
4.Phytochemical Screening
Total Phenolic Contents
Test of Alkaloids
Test of Flavonoids
Test of Protein
Test of Steroids
Test of Glycosides
Test of Tannins
Test of Saponin
Test of Carbohydrate
5. IR of Cinnamomum tamala leaves
extract.
6. IR of Solanum nigrum leaves
extracts.
7. Determination of λmax by UV
Spectroscopy
8. Solubility Studies
9

10. Formulation
1.Formulation Development
 Selection of Methodology
Selection of Process Variables
 Selection of Optimized Formulation
 Formulation of Aluminum Nanoparticles
2.Characterization of Aluminum Nanoparticles
U.V visible absorption of Aluminum nanoparticle extracts.
Particle size and polydispersity index.
Surface morphology
Entrapment efficiency.
 In-Vitro drug Release
Release drug kinetics
3. Antimicrobial Activity
4. Computational and compilation of data
8

11. Methodology
I- Pre-formulation studies
1. Collection of Plant Material
Procured from Aliganj Nursery and S. nigrum collected from Gangadin Khera, Lucknow U. P.
India.
2. Identification and Authentication of Plants
The green leaves was recognized and authenticated by Dr. L.B. Chaudhary, Scientist and Curator
of Herbarium, Plant Diversity and Herbarium Division, NBRI, Lucknow with Ref. no for Solanum
nigrum NBRI/LWG/105169/2019. and Cinnamomum tamala NBRI/LWG/105170/2019.
3. Drying of Plant materials
leaves was cleaned using tap water & dried in shady area at room temperature.
4. Coarse Powder of Plant Leaves
The shady dried leaf of C. tamala & S. nigrum were crushed into coarsest powder and sieved
through sieve number 10 & stored in airtight container for further use.
11

12. II- Preparation of extracts
5. Microwave assisted isolation of Methanolic Extract of Cinnamomum tamala and Solanum
nigrum Leaves
The extraction of C. tamala & S. nigrum leaves were performed by microwave method
6. Preliminary Test of Microwave Assisted methanolic extracts of Cinnamomum tamala &
Solanum nigrum
12
S. No. Parameters
Results
(Cinnamomum tamala)
Results
(Solanum nigrum)
1 Color of extract Dark green Dark green
2 Odor of extract Aromatic and pleasant Aromatic and pleasant
3 Total yield obtained 30g 28.5g

13. 13
S. No Phytochemical screening (Test) Result for
Solanum nigrum
Result for
Cinnamomum tamala
1. Dragendroff’s test (Alkaloid) + +
2. Salkowki test (Steroid) _
+
3. Flavonoid test + +
4. Saponins test + +
5. Gycosides test _ +
6. Tannin test + +
7. Millon’s test (Proteins) _ _
8. Ferric chloride, lead acetate test
(Phenolic compound) + +
9. Carbohydrate test _ +

14. 14
7. Determination of λmax by UV spectroscopy
8. Development of Calibration Curve of Gallic Acid
S. No. Concentration (µg/mL) Absorbance
1 0 0.000
2 1 0.068
3 2 0.121
4 3 0.185
5 4 0.249
6 5 0.307

15. 9. Solubility Studies
15
10. Functional Group Analysis by FTIR Spectroscopy
FTIR Spectroscopy of C. tamala Leaves Extract
FTIR Spectroscopy of S. nigrum Leaves Extract
S. No. Solvents
Solubility of Extracts
Cinnamomum tamala Solanum nigrum
1 Distilled water Soluble Soluble
2 Chloroform Soluble Very Soluble
3 Diluted HCl Poorly soluble Poorly soluble
4 Methanol Freely Soluble Freely Soluble
5 Ethanol Freely Soluble Freely Soluble
6 Phosphate buffer (pH 7.4) Soluble Soluble

16. 11. FORMULATION DEVELOPMENT
Selection of Methodology
Selection of Optimized Condition for Preparation of Aluminium Nanoparticles of Extracts via
Microwave Oven Method
16
Batch
Code
Amount of C. tamal and
S. nigrum Leaves
Extract (mg)
Amount of
Aluminum nitrate
(mg)
Power (w) Time (min)
T1 40 10 560 10
T2 40 15 560 15
T3 40 15 560 20
S1 40 10 560 10
S2 40 15 560 15
S3 40 15 560 20

17. 12. Characterization of Microwave Synthesis of Aluminum Nanoparticles of C. tamala
and S. nigrum Leaves Extract
Batch code Particle size (nm) Zeta potential (mv) PDI
T1 213.2 -25.5 0.618
T2 182.1 -17.3 0.629
T3 141.9 -27.5 0.551
S1 250.0 -21.2 0.515
S2 145.3 -18.1 0.497
S3 125.8 -29.1 0.497
17

18. 13. Experimental Design for preparation of Microwave Assisted Aluminum
Nanoparticles of C. tamala and S. nigrum Leaves Extract
Batches X1 X2 X3
CT1 -1 -1 -1
CT2 -1 -1 +1
CT3 -1 +1 +1
CT4 +1 +1 +1
CT5 +1 +1 -1
CT6 +1 -1 +1
SN1 -1 -1 -1
SN2 -1 -1 +1
SN3 -1 +1 +1
SN4 +1 +1 +1
SN5 +1 +1 -1
SN6 +1 -1 +1
18
22 × 31 Factorial Design for the Optimization of Microwave Assisted Aluminum Nanoparticles
of C. tamala and S. nigrum Leaves Extract

19. 14. Evaluations of Microwave Assisted Aluminium Nanoparticles using C. tamala
& S. nigrum Leaves Extract
S. No. Batch code
Particle size
(nm)
Zeta potential
(mv)
PDI
Entrapment
efficiency (%)
1 CT1 118.7 -27.59 0.243 70
2 CT2 122.58 -18.87 0.447 61
3 CT3 95.3 -30.5 0.397 92.6
4 CT4 92.11 -28.4 0.275 75.2
5 CT5 87.1 -29.1 0.275 69
6 CT6 85.0 -34.4 0.298 86
7 SN1 127.4 -17.8 0.361 62
8 SN2 119.2 -27.91 0.285 68
9 SN3 76.28 -26.1 0.251 79
10 SN4 77.54 -22.0 0.259 86
11 SN5 72.74 -21.4 0.251 81
12 SN6 98.43 -29.5 0.268 90

20. 15. Cumulative Drug Release of Microwave Assisted of Aluminum Nanoparticles
using C. tamala & S. nigrum Leaves Extract
% Cumulative Drugs Release of Microwave Assisted Aluminium nanoparticles using C.
tamala Leaves Extract
Time
(hrs.)
CT1
(µg/cm2)
CT2
(µg/cm2)
CT3
(µg/m2)
CT4
(µg/cm2)
CT5
(µg/cm2)
CT6
(µg/cm2)
0 0 0 0 0 0 0
1 59.02 56.15 56.52 51.44 55.49 56.14
2 60.25 63.25 66.73 63.68 65.43 63.23
3 72.98 70.46 76.16 71.26 72.21 68.74
4 81.26 79.35 83.55 81.49 86.11 76.32
5 89.62 86.89 92.53 89.49 90.86 86.43
6 90.28 92.72 112.45 93.128 102.26 99.12
24 105.125 99.92 121.223 114.86 110.117 121.016
20

21. % Cumulative Drug release of Microwave Assisted Aluminium Nanoparticles of S. Nigrum
Leaves Extract
Time
(hrs.)
SN1
(µg/cm2)
SN2
(µg/cm2)
SN3
(µg/cm2)
SN4
(µg/cm2)
SN5
(µg/cm2)
SN6
(µg/cm2)
0 0 0 0 0 0 0
1 53.02 56.05 51.44 58.52 55.49 59.1
2 62.05 62.45 68.68 62.23 65.43 60.53
3 73.98 70.56 74.26 70.36 72.21 68.24
4 80.26 81.45 81.49 81.15 86.11 78.32
5 90.62 90.89 90.49 85.43 90.86 89.43
6 99.28 99.72 96.128 95.45 99.26 96.12
24 112.125 116.011 114.56 119.023 110.117 121.016
21

22. 16. ANTI MICROBIALACTIVITY
S. No. Batch code Conc.
(µl)
Diameter of zone of inhibition (mm)
S. aureus) P. aeruginosa C. albicans Trichoderma
1 CT1 100 20.4 19.6 19.6 16.6
2 CT2 100 19.8 12.2 20.4 18.7
3 CT3 100 24.6 22.3 21.6 20.3
4 CT4 100 21.5 18.6 1.4 18.6
5 CT5 100 21.2 19.1 16.7 1.2
6 CT6 100 1.1 20.6 19.8 6.9
7 SN1 100 15.5 20.2 18.7 1.9
8 SN2 100 18.8 17.1 17.9 8.5
9 SN3 100 17.9 18.4 18.5 12.9
10 SN4 100 8.7 19.8 19.3 15.8
11 SN5 100 22.6 20.2 20.4 27.6
12 SN6 100 20.8 21.9 22.1 21.7
13 Ciprofloxacin 100 26.3 23.3 - -
14 Amphotericin B 100 - - 23.6 22.06
22

23. Conclusion
The present work was based on the novel, fast, accurate, feasible and convenient technique
was utilized for the formulation of aluminium nanoparticle of Cinnamomum tamala &
Solanum nigrum leaves extract for high yield, good drug loading and increased drug
efficiency etc. aluminium nanoparticles provides faster and prolonged action, increases
product efficiency. Cumulative drug release studies of aluminium nanoparticles of
Cinnamomum tamala & Solanum nigrum leaves extracts showed that aluminium
nanoparticles provide faster action and prolonged activity.
23

24. REFERENCES
1. Agarwal, Happy, Amatullah Nakara, Soumya Menon, Venkat Kumar Shanmugam.
“Eco-friendly synthesis of zinc oxide nanoparticles using Cinnamomum tamala leaf
extract and its promising effect towards the antibacterial activity.” Journal of drug
delivery science and technology 53 (2019): 12-16.
2. Ahmadi, Omid, Hoda, Jafarizadesh, Malmiri and Naeimeh jodeiri. “Ecofriendly
microwave enhanced green synthesis of silver nanoparticles using Aloe vera leaf
extracts and their physio chemical and antibacterial studies.” Green process synthesis,7
(2018):321-240.
3. Aiyalu, Rajasekaran, Arulkumaran, Govindarjan and Arivukkarasu Ramasamy.
“Formulation and Evaluation of topical herbal gel for the treatment of Arthritis in
animal model.” Brazillian journal of pharmaceutical sciences, 52 (2016):493-507.
4. Algabri, o Sumia, Awatf m.Abadi, Mohmoud A. Shiba and Almonder H. Salem. “Bay
leaves have antimicrobial and antioxidant activities.” Journal of Pathogens research,1
(2018):1-5
5. Andrews, M. Jennifer. “Determination of Minimum inhibitory concentrations.” Journal
of Antimicrobial chemotherapy,48 (2001):5-16
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25. 6. Kapoor, IPS, Bandana Singh, Gurdeep Singh, Valery Isidorov and Lech Szczepaniak,
“Chemistry, antimicrobial and antioxidant potentials of Cinnamomum tamala Nees and
Eberm. Essential oil and oleoresins.” Natural product Radiance, 8 (2009):106-116.
7. Nahar, Kamrun, Shahin, Aziz, Muhammad, Shahriar, Bashar, Md., Ahsanul, Haque and
Sharif Md., Ali, Reza. “Synthesis and characterization of Silver nanoparticles from
Cinnamomum tamala leaf extracts and its antibacterial potential.” Int. j. nano. Dimens,
11 (2020):88-98.
8. Wang, Rui, Ruijiang wang, Bao Yang. “Extraction of essential oils from five cinnamon
leaves and identification of their volatile compound compositions.” Innovative food
science and emerging tehnologies,10 (2009): 289-292.
9. Xin, Yong, Pang and Xu, jin Cha, Bao. “Aluminium Oxide nanoparticles prepared by
Water- in -oil Microemulsions.” The Royal Society of Chemical, 12 (2002):3699-3704
10.Yie w. chein. “Novel Drug delivery system” 2nd edition Vol- 2 Marcel Dekker press-
United states of America. (2010):786-792
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