KIMIA BAHAN ALAM TERESTERIAL

ISOLATION AND BIOACTIVE

NATURAL PRODUCTS
Mutista Hafshah, M.Si
Jumat, 24 Mei 2019
Overview
Pigment isolated from Streptomyces K-4b

Eugenol isolated from clove bud (Syzygium aromaticum)

Terpenoids isolated from Azadirachta indica

Bioactive natural products

 Phytochemical test

Secondary Metabolites Solvent test Positive Results

Mayer: white sediment
Mayer, Dragendorf, and Dragendorf: reddish yellow
Alkaloid
Wagner solvents sediment
Wagner: reddish brown sediment
Flavonoid HCl 2N + 0,2 g Mg Magenta solution in 3 minutes

Saponin Distillled water + HCl Shaked = bubbles

Tannin FeCl3 1% Greenish black or dark blue

Acetic acid + sulfuric Triterpenoid : Violet
Triterpenoid and steroid
acid Steroid : greenish blue
 Pigment isolated
from Streptomyces
K-4b
 Pigment isolated from Streptomyces K-4b

Extraction Characterization
• Incubation and • Thin layer
centrifugation • Sonication chromatograph • UV-Vis
process • column
• Soxhletation Spectrophotomet
chromatography er
• FTIR
Production Purification • NMR
 Pigment producted from Streptomyces K-4b

Incubation Centrifugation
process process

Isolate Streptomyces K-4b

Micellium of Streptomyces K-
4B
Inoculum of Streptomyces K-4B
 Instruments

centrifugator Sonicator

centrifugator
 Pigment extracted from Streptomyces K-4b

Soxhletation
process
Sonication
process

Micellium + 50 mL ethanol 96%

Evaporation
process
 Instruments

Soxhlet Evaporator
 Pigment
Purification

Thin layer Column Chromatography

Chromatography
Eugenol isolated
from clove bud
(Syzygium
aromaticum)
 Eugenol Isolation
• Clove oil extraction • GC-MS
by steam distillation
• Eugenol Isolation by
Liquid-Liquid
Extraction

Clove oil Extraction Characterization

Eugenol isolated from clove bud (Syzygium

aromaticum)
Clove oil Extraction

Ground clove

GC-MS
Analysis

Clove oil
Eugenol Acid Base Reaction
Isolation
Eugenol Isolation

NaCl and
H 2O

Na-eugenolat
and H2O

Eugenol

Low eugenol GC-MS

clove oil
Analysis
BIOACTIVE
NATURAL
PRODUCTS
 In-vitro Anti-microbial activity

Agar diffusion methods:

Notes:
a. Disk diffusion method of Microbial
extract using C. albicans
b. Agar well diffusion method of essential
oil using Aspergillus niger

Balouiri, M., Sadiki, M. and Ibnsouda, S.K., 2016. Methods for in vitro evaluating antimicrobial activity: A review. Journal
of pharmaceutical analysis, 6(2), pp.71-79.
Dilusi Cair
 TLC Bioautogafi

Patil, N.N., Waghmode, M.S., Gaikwad, P.S., Gajbhai, M.H. and Bankar, A.V., 2013. Bioautography guided screening of
antimicrobial compounds produced by microbispora V2. Int. Res. J. Biological Sci, 2(2), pp.65-68.
Isolasi senyawa antibakteri dari ekstrak Anthocephalus indicus A. Rich. dianalisis
menggunakan metode TLC-bioautography. Bioautography termasuk dalam metode
skrining mikrobiologi yang biasa digunakan untuk mendeteksi aktivitas antibakteri dari
senyawa aktif tanaman. The fraction of fruit extract was loaded on the TLC plates for the
separation of compounds responsible for antibacterial activity. Preactivated TLC plate
coated with silica gel G 60 F254 (Merck) was used as stationary phase. Ethyl acetate:
ethanol: water (8: 2: 0.2) was used as mobile phase [19]. In the agar-overlay
bioautography method, the TLC plate with separated compounds is first placed on the
surface of Nutrient agar medium in the petri plates and then covered with Nutrient agar
medium seeded with the test organisms. The plates were then incubated at 37°C for 24
hours. The zone of inhibition formed due to antimicrobial agent on the TLC plates was
visualized using triphenyl tetrazolium chloride reagent [20].
 In-vivo Anti-microbial activity

Punica
granatum

Importance of PGPE against S. typhimurium infection

Choi, J.G., Kang, O.H., Lee, Y.S., Chae, H.S., Oh, Y.C., Brice, O.O., Kim, M.S., Sohn, D.H., Kim, H.S., Park, H. and Shin,
D.W., 2011. In vitro and in vivo antibacterial activity of Punica granatum peel ethanol extract against Salmonella. Evidence-
Based Complementary and Alternative Medicine, 2011
 In-Vitro Anti-diabetic activity

α-amylase inhibition
Measuring the
Starch
+ Plant extract absorbance
was recorded
at 540 nm
DNS Color reagent
 In-Vivo Anti-diabetic activity

Plant extract Streptozotocin-induced

Analysis of blood
glucose levels
 Starch

Streptozotocin
 In-vitro Anti-oksidant activity
DPPH
Scavenged (%)
Antioxidant Absorbance measurement by
Sample
activity by DPPH spectrophotometer UV-Vis (λ 517 nm)
IC50

DPPH in methanol + Sample DPPH in Ethanol +

with different concentration Methanol

DPPH in Methanol
DPPH Scavenged (%)

Negative S1 S2 S3 S4 Positive

Control Control
The Maximum Wavelength of DPPH DPPH Scavenging (%) of Eugenol
80.00% 72.50%
70.00%
f(x) = 0.05962818370357 x + 0.13716409436793
R² = 0.99670036713291 58.78%
60.00%

DPPH Scavenging (%)

50.00% 42.85%
40.00% 32.31%
30.00%
18.66%
20.00%
10.00%
0.00%
0 1 2 3 4 5 6 7 8 9 10
Eugenol Concentration (ppm)

IC50 = 6.087 ±
0,002 μg/mL
 In-vivo Anti-oksidant activity

Oxidative
Plant extract + CCl4
toxicity

MDA, SOD, CAT, and

GSH levels measured

Anti-oxidant reduced elevated

MDA and increased SOD, CAT
Coptis Chinensis and GSH levels

Ban, X., Huang, B., He, J., Chen, Y., Zeng, H., Han, L. and Wang, Y., 2011. In vitro and in vivo antioxidant properties of extracts
from Coptis chinensis inflorescence. Plant foods for human nutrition, 66(2), pp.175-180
 In-vitro Anti-cancer activity

Inhibition of colony formation of A-549

lung cancer cells with A. nitida
treatment
Notes:
a. Control mouse
b. ANL 5 µg/mL treated cells
c. ANL 10 µg/mL treated cells
d. ANB 5 µg/mL treated cells
e. ANB 10 µg/mL treated cells

Sajid, M., Yan, C., Li, D., Merugu, S.B., Negi, H. and Khan, M.R., 2019. Alnus nitida
Potent anti-cancer activity of Alnus nitida against lung cancer cells; in vitro
and in vivo studies. Biomedicine & Pharmacotherapy, 110, pp.254-264.
 In-vivo Anti-cancer activity

Effect of ANL and ANB on lung surface in xenograft model of mouse

Notes:
a. Control mouse
b. ANL 10 mg/kg bw
c. ANL 20 mg/kg bw Sajid, M., Yan, C., Li, D., Merugu, S.B., Negi, H. and Khan, M.R., 2019.
d. ANB 10 mg/kg bw Potent anti-cancer activity of Alnus nitida against lung cancer cells; in vitro
and in vivo studies. Biomedicine & Pharmacotherapy, 110, pp.254-264.
e. ANB 20 mg/kg bw
 References
(1) A’yun, Q., 2015. Analisis Fitokimia Daun Pepaya (Carica papaya L.) di Balai Penelitian Tanaman Aneka
Kacang dan Umbi, Kendalpayak, Malang. In Seminar Nasional Konservasi dan Pemanfaatan Sumber Daya Alam
2015. Sebelas Maret University.

(2) Balouiri, M., Sadiki, M. and Ibnsouda, S.K., 2016. Methods for in vitro evaluating antimicrobial activity: A
review. Journal of pharmaceutical analysis, 6(2), pp.71-79.

(3)Ban, X., Huang, B., He, J., Chen, Y., Zeng, H., Han, L. and Wang, Y., 2011. In vitro and in vivo antioxidant properties
of extracts from Coptis chinensis inflorescence. Plant foods for human nutrition, 66(2), pp.175-180.

(4) Choi, J.G., Kang, O.H., Lee, Y.S., Chae, H.S., Oh, Y.C., Brice, O.O., Kim, M.S., Sohn, D.H., Kim, H.S., Park, H.
and Shin, D.W., 2011. In vitro and in vivo antibacterial activity of Punica granatum peel ethanol extract against
Salmonella. Evidence-Based Complementary and Alternative Medicine, 2011.

(5) Passos, M.D.S., de Carvalho Junior, A.R., Boeno, S.I., das Virgens, L.D.L.G., Calixto, S.D., Ventura, T.L.B.,
Lassounskaia, E., Braz-Filho, R. and Vieira, I.J.C., 2019. Terpenoids isolated from Azadirachta indica roots and
biological activities. Revista Brasileira de Farmacognosia, 29(1), pp.40-45.

(6) Sajid, M., Yan, C., Li, D., Merugu, S.B., Negi, H. and Khan, M.R., 2019. Potent anti-cancer activity of Alnus
nitida against lung cancer cells; in vitro and in vivo studies. Biomedicine & Pharmacotherapy, 110, pp.254-264.
Ekstraksi pelarut
Destilasi uap
TL
C
Kolom Kromatografi
TLC-
preparative
Cyclograph
UTS
Buat Resume
Dikumpulkan Jumat, 18 Oktober 2019 Pukul 16.00

1. Cari 5 jurnal, terdiri dari 2 jurnal internasional dan 3 jurnal nasional

(dilampirkan) mengenai metode uji bioaktivitas
2. Usahakan masing-masing jurnal metode uji bioaktivitasnya berbeda satu
sama lain.
3. Buat resume menggunakan kertas double folio ditulis tangan (dengan
tulisan yang rapi dan jelas).
4. Isi resume:

• Sumber bahan alam (nama tumbuhan dan nama latinnya)

• Metode Uji Bioaktivitas
• Prinsip dasar Metode Uji Bioaktivitas
• Cara Kerja (dibuat tabel)
• Hasil Analisis
1. Antibakteri secara in Vitro
2. Antibakteri secara in Vivo
3. Antioksidan secara in Vitro
4. Antioksidan secara in Vivo
5. Antidiabetes secara in Vitro
6. Antidiabetes secara in Vivo
7. Anticancer secara in Vitro
8. Anticancer secara in Vivo
9. Antijamur secara in Vitro dan in Vivo