ANTIMICROBIAL ACTIVITY OF

ENCAPSULATED MORUS SP.

(MULBERRY)
LEAVE EXTRACTS
BY: NUR SYUHADA ADNAN
SUPERVISOR: DR. CHE WAN SHARIFAH ROBIAH MOHAMAD
CO. SUPERVISOR: DR KHAIROL FARIHAN KASIM
 INTRODUCTION
1. Infectious diseases are one of the major public health problem (57 million) [1]
• is caused by organisms known as pathogens Fauci et al. (2005)
• synthetic antibiotics effect is limited by the toxic effect and multi drug resistance [22]

2. Plants contain numerous organic compound

• these compounds have anticancer, antibacterial, analgesic, anti-inflammatory, antitumor,
antiviral [4].
• inhibit the growth of bacteria and fungi while showing less toxic effect on the patient [23]
Cont…
1. Morus is a type of flowering plants in the family of
Moraceae.
2. A reservoir of antioxidant.
3. High potential to fight infectious disease.

1. E. coli, P. aeruginosa and S. aureus. Sharecare Inc

2. Cause gastritis by changing stomach normal pH.

1. Important to act as a physical barrier.

2. Encapsulation increases the efficiency of ingredients
3. Release ingredients at optimal time.
PROBLEM STATEMENTS

1. Gastritis is pandemic and 2. Mulberry leaves research does not

worldwide. provide optimal time absorption.

3. Synthetic gastritis medicine effect is

limited by toxic effect and multi drug
resistance.
OBJECTIVES
1. To analyze the phytochemicals composition of encapsulated
mulberry leaves extract by evaluating the total phenolic
content (TPC), total flavonoid content (TFC) and antioxidant
activity (AOA).
2. To determine the antimicrobial activity of encapsulated
mulberry leaves extracts.
3. To correlate and characterize the properties of the
encapsulated mulberry leaves extracts.
SCOPE AND LIMITATION
1. Collection and extraction of mulberry leave by methanol and
ethanol
2. Encapsulation of mulberry extract
3. Phytochemical test for TPC, TFC and AOA.
4. Antimicrobial test using E. coli, P. aeruginosa and S. aureus.
5. Characterization of best encapsulated mulberry by using
FESEM.
6. Correlation and statistical analysis by SPSS AND Minitab
LITERATURE REVIEW
1) Plant sample
2) Extraction method
3) Extraction solvent
4) Encapsulation
5) Antimicrobial activity test
LITERATURE REVIEW
Useful both in the
treatment of
diabetes and in its
prevention [16]

Antibacteria
[2,9]
Mulberry leaves

High in Has anti-

antioxidant. inflammatory
More than 80% effects [2,9]
[2,7,12,13] Reduce total
cholesterol [16]
 MULBERRY LEAVES
Sample
Author Title Fresh Dried
TPC TFC) AOA TPC TFC AOA
Sánchez-Salcedo, E. M., Mena, P., (Poly)phenolic compounds and antioxidant activity 16.13 13.15
García-Viguera, C., Hernández, F., of white (Morus alba) and black (Morus nigra)
& Martínez, J. J. (2015). mulberry leaves
Edwin H. Mena* , Tao Liu, Xianyan Effect of Superfine Grinding on the Phytochemicals 52.30 38.03 73.31
Liao, Tosin M. Olajide, Junyi Huang and Antioxidant Activities of Mulberry Leaves
(2016)
Ching-Hui Chang a,b, Hsing-Yu Lin Comparisons on the antioxidant properties of fresh, 38 4.8 97 43 9.0 99
b, Chi-Yue Chang b, Yung-Chuan Liu freeze-dried and hot-air-dried tomatoes

Zhishen, J., Mengcheng, T., & The determination of flavonoid contents in mulberry 15.3 11.5
Jianming, W. (1999). and their scavenging effects on superoxide radicals.

Learning outcome:
1. Dried sample shows an average high reading of phytochemical compound.
2. Fresh sample shows high but inconsistant reading of phytochemical compound.
3. Therefore, the best sample for extraction process is dried leaves
Extraction method

1. Soxhlet extractor (invented in 1879 by Franz von Soxhlet)

2. Maceration (first described by Jean-Martin Charcot in 1877)
3. Ultrasound extraction
 Method
Soxhlet Maceration Ultrasound
Author Title
TPC (mg TFC (mg AOA (%) TPC (mg TFC (mg AOA (%) TPC (mg TFC (mg AOA (%)
GAE/g) QE/g) GAE/g) QE/g) GAE/g) QE/g)

Edwin H. Mena, Effect 41.61±1. 22.74±0. 55.9±1.0

Tao Liu, Xianyan of Superfine 30 99
Liao, Tosin M. Grinding on the
Olajide, Junyi Phytochemicals and
Huang. (2016) Antioxidant
Activities of
Mulberry Leaves

Brijesh R., Evaluation of NR NR 34.63±0.5

Randhir K. , antioxidant activity, 33.74±1.3
Neeraj V. , Swati phytochemical
M. , Pranab L. P. constituents and
and Venkatesh Anti-diabetic
Kumar R. properties of C763
mulberry leaves
Zhishen, J., The determination NR 20.7 46.5
Mengcheng, T., & of flavonoid
Jianming, W. contents in mulberry
(1999) and their scavenging
effects on
superoxide radicals.
 Method
Soxhlet Maceration Ultrasound
Author Title
TPC (mg TFC (mg AOA (%) TPC (mg TFC (mg AOA (%) TPC (mg TFC (mg AOA (%)
GAE/g) QE/g) GAE/g) QE/g) GAE/g) QE/g)

Zhang, D.-Y., Ultrasound NR NR 15.59-

Wan, Y., Xu, J.-Y., extraction of 42.35
Wu, G.-H., Li, L., polysaccharides
& Yao, X.-H. from mulberry
(2016). leaves and their
effect 9 on
enhancing
antioxidant activity
Saeedah A. D., Antioxidant 7.10 – 9.32 NR 36.92 –
Asna Urooj properties of various 81.80
solvent extracts of
Mulberry Leaves
Shoib A. Baba, Determination of 45±1.7 35.5±2.2 NR
Shahid A. Malik TPC , TFC and
antimicrobial activity
of Arisaema Jacque
monti Blume

Learning outcome:
1. Ultrasound assisted extraction shows lowest AOA reading.
2. Maceration shows the highest reading than soxhlet extraction
Learning outcomes:
Extraction method
Maceration / Decoction Method Soxhlet Extraction Method

• Take longer time • Shorter extraction time

• Each cycle material to be process get
used solvent
• Come in contact with impure solid
products
• Extracts produced are not pure
• Each cycle material to be process get
fresh solvent
• Does not come in contact with impure
solid products
• Extracts produced are pure

* To ensure antimicrobial activity against selected bacterial went successfully,

soxhlet extraction is selected.
 Extraction solvent
Compon Nicaragua India Niger
ents
Water Methanol Ethanol Water Methanol Ethanol Water Methanol Ethanol
extracts extract extracts extracts extract extracts extracts extract extracts
TPC 7.43 ± 12.33 ± 11.04 ± 5.25 ± 8.87 ± 8.11 ± 6.83 ± 9.76 ± 8.78 ±
0.09 0.46 0.42 0.34 0.07 0.06 0.30 0.34 0.31
TFC 10.83 ± 14.07 ± 10.14 ± 3.26 ± 5.76 ± 5.92 ± 7.32 ± 10.19 ± 9.01 ±
0.15 0.83 0.47 0.24 0.10 0.16 0.46 0.18 0.12
Values are means of triplicate determination ± standard deviation
Perumal Siddhuraju And Klaus Becker. (2003) [17]
Learning outcomes:
1. Water shows the lowest reading.
2. Methanol shows the highest reading.
3. Therefore Methanol and ethanol is selected as the extraction solvent to
make comparison for statistical analysis.
Encapsulation
Author Title Size Findings
Anubhav Nagal, Nanoparticles in 10-1000nm • 100 nm nanoparticles had a 2.5 fold greater uptake than 1 μm
Rajeev K Singlab Different Delivery microparticles, and 6 fold greater uptake than 10 μm
(2013) Systems: A Brief microparticles
Review • The paracellular route of absorption of nanoparticles utilises
less than 1% of mucosal surface area.
• Encapsulation can increases the efficiency of active
ingredients and protect from physical barrier
Wani, T. A., Shah, Suitability of Different 75-750nm • Insulin that is encapsulated in nanoparticles (NP) is less
A. G., Wani, S. M., Food Grade Materials sensitive to enzymatic degradation
Wani, I. A., for the Encapsulation • NP are useful for the effective intestinal absorption of insulin
Masoodi, F. A., of Some Functional
Nissar, N., & Foods Well Reported
Shagoo, M. A. for Their Advantages
(2015). and Susceptibility

Learning outcomes:
• Encapsulation increase the efficiency of active ingredients
• Nano particle provide better absorption in intestinal area (GI tract)
Antimicrobial Activity
Bacteria

Author Title E. Coli P. S. Aureus

Aeruginosa

M. O. Omidiran, R. A. Baiyewu, I. Phytochemical Analysis, Nutritional Composition and 8mm 8mm 10mm
T. Toyinbo, O. J. Adewumi, E. A. Antimicrobial Activities of White Mulberry (Morus Alba) 900µg/ml 900µg/ml 800µg/ml
Adekunle
Shoib A. Baba, Shahid A. Malik Determination of TPC , TFC and antimicrobial activity of 340 µg/ml NR 370µg/ml
Arisaema Jacque monti Blume
James R., Malcolm K., Dariel. B., Soxhlet Ethanol Extraction to Produce and Test Plant Material 16mm NR 9mm
Joanna V. (Essential Oil) for Antimicrobial Activity

Chaitali Ravi Niratker*, Preeti Antimicrobial activity of leaf extract of Morus indica (Mulberry) 0mm 2mm 12-15mm
and Malti from Chhattisgarh

Learning outcomes:
1. Less inhibited bacteria is E.coli
2. Most inhibited bacteria is S.Aureus
Methodology Start

Sample preparation

Extraction

Encapsulation

Antimicrobial screening Phytochemical test

Correlation and characterization

End
 1. Sample preparation

1. Plant collection 2. Washing (tap water) 3. Rinsing (distilled water) 4. Drying at room 5. Cutting into small
temperature pieces

6. Drying in oven 7. Blending/grinding 8. Sieve (fine powder)

2. Extraction 3. Encapsulation 4. Freeze dry 5. Phytochemical test

Folin-Coiocalteu colorimetric method

Dialysis

Soxhlet

Aluminium chloride colometric method

FESEM

DPPH method

UV-VIS
6. Antimicrobial 7. Correlation and
activity characterization

Minitab, SPSS, PAC

Broth dilution method

PLANNING (GANTT CHART)
EXPECTED RESULT
Encapsulation by Dialysis Tubing
• Microcapsule size of encapsulated mulberry leaves extracts.
• The particle size is uniform.

Phytochemical Analysis
• Methanol extracts will give a higher TPC, TFC AND AOA reading compared to
ethanol.

Antimicrobial Activity
• MC encapsuled extract will give a higher percentage of bacteria inhibition
• S. Aureus will show least inhibition percentage.
REFERENCE
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2. Arabshahi-Delouee, S., & Urooj, A. (2007). Antioxidant properties of various solvent extracts of
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Journal of King Saud University - Science, 27(3), 224–232.
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Cont...
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(n.d.). Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus Attachment Patterns on
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(2015). Analysis and characterisation of phytochemicals in mulberry (Morus alba L.) fruits grown in
Vojvodina, North Serbia. Food Chemistry, 171, 128–136.
8. Park, S., Kim, J., & Kim, Y. (2012). Mulberry Leaf Extract Inhibits Cancer Cell Stemness in
Neuroblastoma. Nutrition and Cancer, 64(6), 889–898.
9. Sánchez-Salcedo, E. M., Mena, P., García-Viguera, C., Hernández, F., & Martínez, J. J. (2015).
(Poly)phenolic compounds and antioxidant activity of white (Morus alba) and black (Morus nigra)
mulberry leaves: Their potential for new products rich in phytochemicals. Journal of Functional Foods,
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and their scavenging effects on superoxide radicals. Food Chemistry, 64(4), 555–559.
Cont…
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(2011). Encapsulation of polyphenolic antioxidants from medicinal plant extracts in alginate–chitosan system
enhanced with ascorbic acid by electrostatic extrusion. Food Research International, 44(4), 1094–1101.
13. Dib, M. E. A., Allali, H., Bendiabdellah, A., Meliani, N., & Tabti, B. (2013). Antimicrobial activity and
phytochemical screening of Arbutus unedo L. Journal of Saudi Chemical Society, 17(4), 381–385.
14. Gryn-Rynko, A., Bazylak, G., & Olszewska-Slonina, D. (2016). New potential phytotherapeutics obtained from
white mulberry (Morus alba L.) leaves. Biomedicine & Pharmacotherapy, 84, 628–636.
15. Iqbal, E., Salim, K. A., & Lim, L. B. L. (2015). Phytochemical screening, total phenolics and antioxidant
activities of bark and leaf extracts of Goniothalamus velutinus (Airy Shaw) from Brunei Darussalam. Journal of
King Saud University - Science, 27(3), 224–232.
16. Mudra, M., Ercan-Fang, N., Zhong, L., Furne, J., & Levitt, M. (2007). Influence of mulberry leaf extract on the
blood glucose and breath hydrogen response to ingestion of 75 g sucrose by type 2 diabetic and control
subjects. Diabetes Care, 30(5), 1272–1274.
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Phenolic Constituents From Three Different Agroclimatic Origins Of Drumstick Tree (Moringa Oleifera Lam.)
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Pathogenic Bacteria. Pharmaceuticals, 6(12), 1451–1474.
Cont…
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mulberry leaves and their effect on enhancing antioxidant activity. Carbohydrate Polymers, 137, 473–479.
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Additional information
Type of sample
Fresh Sample Dried Sample
• Easy to obtain and less procedure • Hard to obtain as it need to undergoes more
procedure
• Not compatible with chemical solvent • Compatible with chemical solvent
• Change according to climate, weather • No change according to climate, weather

• Moisture content in plant varies • Moisture content in plant varies does not varies

• Undergoes further enzymatic or metabolic alteration • No further enzymatic or metabolic alteration

• Gives more error • Dry weight gives less error

Therefore, the best sample for extraction process is dried leaves

 Antimicrobial Activity Test
• Brantner and Grein et al (1994)
1. Agar disc dilution method • Ali et al. (1997)
• Lysis (inhibition) zone diameter
• Bacterial culture adjusted to McFarland turbidity standards and inoculated onto 15cm petri
dish.
• Make 5 small holes in the agar to put bacterias and controls.
• Bacterial cultures were then incubated at 37c for 24h.
• Antimicrobial activity was determine by measurement of inhibition zone around agar disc.

2. Broth dilution method • Carson et al. (1995)

• Minumum inhibitory Concentration (MIC) • Iwaki et al. (2006)
• Serial tube dilution technique was used to determine MIC • Khan et al. (2007)
• A few drops of HCl were added into mixture until pH 4
• Test tubes containing a mixture of bacteria and the extracts were incubated at 37 ºC for 24 h.
• The antimicrobial activity was noted by visual turbidity of test tube before and after incubation.
The mixture was measured at 600 nm after 24 h.
Phytochemical test
• TPC
• The Folin-Coiocalteu colorimetric method was used to determine TPC in mulberry extracts.
• 0.1 mL of sample extracts was added into 0.2 mL of Folin-Ciocalteu and 8 mL of distilled water.
• The mixtures were mixed thoroughly and incubate for 3 min. Then, 1 mL of Na2CO3 (20%) was
added and it was kept in dark place for 30 min.
• The mixture was measured at 765 nm in a UV-VIS.
• The TPC was expressed as mg gallic acid equivalents (GAE)/g extract.

• TFC
• The total of flavonoids in mulberry extracts were determined using aluminum chloride
colorimetric method. 0.5 mL mulberry extracts was taken and added with 2 mL of distilled
water.
• Then, 0.15 mL of NaNO3 (50%) was added into the mixture and incubate for 5 min.
• 0.15 mL of AlCl3 was added and the absorbance was recorded at 415 nm after 15 min of
incubation.
• The TFC was expressed as mg quercetin equivalent (QE)/g of sample.
• AOA
• The antioxidant activity was determined using DPPH method.
• 200 μL of mulberry extracts was mixed with 200 μL of 60 μM DPPH.
• The mixture was incubated for 30 min in dark condition.
• Initially, absorption of blank sample containing same amount of ethanol and DPPH
solution was prepared and measured as a control.
• After that, the solution was measured using UV-VIS at 517 nm.
• The experiment was carried out in triplicates. The percentage inhibition was calculated
using equation below:
Additional information
Antimicrobial activity test
Agar Disc Dilution Method Broth Dilution Method

• Takes a few day to see the result • Faster result

• Not suitable for solid sample • Suitable for solid sample

• Observe lysis zone • Observe the concentration of solution

Literature review
• Methylcellulose and ethylcellulose for encapsulation
• has extensively been used for microencapsulation due to its many versatile properties such as [25]:
1. white to light tan odorless and tasteless powder or granular substance;
2. melting point range 240c-255c;
3. water insoluble but soluble in many organic solvents such as alcohol, ether, ketone and ester;
4. biocompatible
5. nonbiodegradable, thus used in oral formulation only;
6. stable against light, heat, oxygen and wetness and chemicals;
7. non-toxic;
8. non-irritant;
9. ability to absorb pressure and hence protect the coating from fracture during compression.
10. water insoluble
Extraction solvent
Test for Chloroform Methanol Ethanol
Alkaloids -- -- --
Carbohydrates and -- ++ ++
- -: Absent ++: Present
glycosides
Phytosterols -- ++ ++
Fixed oils and fats -- -- --
Phenolic -- ++ ++
Saponins -- -- --
Flavonoids -- ++ ++
Proteins and ++ ++ ++
Amino acids
Gums and -- -- -- M.P. Raghavendra
mucilage (2005) [26]
Volatile oils -- ++ ++