This document describes the methodology used in a project involving the extraction and isolation of compounds from the plant Andrographis paniculata. Key steps included: 1) Collecting and preparing the plant material, then extracting it using methanol and water to obtain crude extracts. 2) Isolating compounds from the crude extracts using chromatographic techniques like column chromatography and centrifugal thin layer chromatography. 3) Characterizing the isolated pure compounds using various analytical instruments and spectroscopy techniques like NMR, IR, MS, UV-Vis and polarimetry. Two pure compounds, coded KZY001 and KZY002, were isolated and identified as andrographolide and 14-deoxy-11,12-

1. 53
CHAPTER 3
METHODOLOGY
3.1 Introduction
In this chapter, we will be discussing the use of chemicals, materials and
instruments in completing this project. The detailed report on the preparation,
parameter, and the conditions for each method used were discussed.
3.2 Apparatus, Materials, Chemicals and Instruments
The main organic solvents and chemicals were purchased from commercial
chemical suppliers and are used without any further purification. Organic
solvents and chemicals used in this project were listed in Table 3.1 and Table
3.2, with purity and supplier’s name provided.
The experimental instruments and analytical instruments used for
characterization and structural elucidation of compounds were listed in Table
3.3. Other consumables, glassware, and apparatus were obtained from the
Department of Chemistry, Universiti Tunku Abdul Rahman, Kampar campus.

2. 54
Table 3.1:Sources and purity of organic solvents used in the project
Solvent Purity / (Grade) Brand/ Company
Chloroform, CHCl3 (AR) QRёC
D-Chlofoform, CDCl3 99.8% MERCK
Ethyl acetate, CH3COOC2H5 (AR) Fisher Scientific
n-Hexane, C6H6 (AR) QRёC
Methanol, CH3OH (AR), (LCMS) Fisher Scientific
D-Methanol, CD3OD 99.8% MERCK
Pyridine, C5H5N (AR) Fisher Scientific
(AR) analytical reagent grade
(LCMS) stands for liquid chromatography/ mass spectrometry grade
Table 3.2: Sources of chemicals used in the project
Chemical Brand/ Company
Anhydrous sodium sulphate, Na2SO4 SYSTERM
Silica gel for C.C (200 – 400 mesh) R&M Chemicals
Silica Gel 60 PF254 Containing Gypsum MERCK
TLC Silica Gel 60 F254 MERCK
TLC Silica Gel RP-18 MERCK
Table 3.3: List of instruments used in the project and its manufacturer
Instrument Manufacturer
Accurate-Mass Q-TOF LC/MS model G6520B Agilent Technologies
HALO SB-10 UV-VIS Single Beam
Spectrophotometer
Dynamica
JNM ECP 400NMR Spectrophotometer JOEL
Rotatory Evaporator Buchi
P-2000 Polarimeter JASCO
Stuart SMP10 Melting Point Instrument BioCote
Spectrum RX1 IR Spectrometer PerkinElmer

3. 55
3.3 Plant Material Collection
The aerial part of Andrographis paniculata was collected along the roadside of
the residential area in Taman Perwira, Pulau Pinang (Latitude: 5.2966510000
Longitude: 100.4743271000). The criteria for the sample collection was based
on the maturity of the plant (plants that are too young and too old were not
being considered) and the condition of the plant itself (free from pest invasion
and are in healthy condition). The plant was first identified by matching its
morphology with an authenticated reference material, and was collected only if
there is a 100% matching (Bucar, Wube and Schmid, 2013).
3.4 Preparation of Plant Material
The collected sample was washed briefly with water before immediately
subjected to sun drying for 7 days, in order to facilitate the removal of water.
Care was taken to prevent any contact of water, e.g., rainwater to the sample
during the dehydrating process. The sample was then snapped, crushed into
smaller pieces before subjected to grinding. The grinded powder obtained was
weighed and found to be 1.37 kg.

4. 56
3.5 Extraction from Raw Plant Materials
The sample powder was divided into two 5000 ml conical flask using a relative
large funnel with large opening to allow a fast, efficient flow of sample powder.
MeOH was poured into the two 5000 ml conical flask at the volume ratio of
2:1 to the volume occupied by the sample powder in the flask. The opening of
the flasks were then sealed using aluminum foils. Each soaking was allowed to
take place for 5 days and the flasks were occasionally shook manually during
the 5 day extraction so as to increase the efficiency of solvent extraction. The
MeOH extracts were drawn out after 5 days and were later concentrated in
vacuo using rotatory evaporator to obtain crude product. The soaking process
was repeated for 7 times with TLC monitoring (Refer to 3.6.1). The MeOH
crude product for each extraction was mixed and weighed after pump-drying.
The MeOH crude product was found to be 355.27 g.
The residual plant material waslater subjected to distilled H2O soaking
following the similar procedures in MeOH soaking. Reversed phase TLC was
used for monitoring and the extraction was done 6 times. H2O extract crude
product with the weight of 96.07 g was obtained after freeze drying and kept in
desiccator to prevent absorption of water.
To ease the isolation process, the crude MeOH extract was partitioned and
extracted successively with hexane, chloroform and ethyl acetate. The
extraction was done twice (1 L of extracting solvent each time) in a 5000 ml
separating funnel for each solvent to increase the efficiency of the extraction.

5. 57
Each partition solvent extract, which is the organic layer in the separating
funnel, was drawn out and concentrated in vacuo to give separate crude, from
each solvent. The weighing of each crude product was done after being pump-
dried. Aqueous layer from the partition extraction were drawn and kept freeze,
ready for dehydration via freeze drying. Observed coarse brown solid was
subjected to further filtration which will be explained in depth in Chapter 4.
Figure 3.1 shows the mapping of work done on the extraction of the aerial part
of the plant.

6. 58
Dried, grinded plant material
(1.37 kg)
MeOH soaking for 7 times
In vacuo evaporation using
rotatory evaporator
Collection of crude
product (355.27 g)
Partition extraction twice using Hex, EA, and CHCl3
(1 L of solvent used for each extraction)
Hex partitioned
extract
Collection of Hex partitioned
crude (36.85 g) after in vacuo
evaporation via rotatory
evaporator
EA partitioned
extract
Collection of EA partitioned
crude (49.66 g) after in
vacuo evaporation via
rotatory evaporator
CHCl3 partitioned
extract
Collection of CHCl3 partitioned crude
(49.66 g) after in vacuo evaporation
via rotatory evaporator
Aqueous extract,
ready for freeze
drying
Brown solids (27.87 g) sendimented at the
bottom of the flask
Residual plant material subjected to
H2O soaking for another 6 times
Collection of crude after
freeze drying (96.07 g)
Figure 3.1: Extraction flowchart of the axial part of Andrographis paniculata.

7. 59
3.6 Separation and Isolation of Compounds from Crude Product
Isolation and purification of pure compounds from a mixture were done
extensively throughout this project. The chromatographic methods used were
CC and CTLC. Application of each method in the separation process is
discussed in this section.
3.6.1 Thin-Layer Chromatography (TLC)
TLC is used routinely for the monitoring of the availability of compounds
during extraction process and the choice of appropriate solvent system for the
isolation purposes in CC and CTLC. The polarity of the solvent affects the
choice of solvent and the separation process, thus an appropriate modifications
on the solvent system are intricately tested with TLC. The polarity of the
solvent system can be either increased or decreased, depending on the
chromatographic spots which can be visualized using UV lamp and iodine
staining. A solvent system that induces a better separation of the compounds
on the TLC plate is preferred.TLC Silica Gel 60 F254 plate was used for normal
phase monitoring whileTLC Silica Gel RP-18 plate was used for reversed
phase monitoring.

8. 60
3.6.2 Column Chromatography (CC)
Due to the time limit that was provided for this project, only CHCl3 crude was
being subjected for isolation and collection of pure compounds. The other
crude products were kept for further studies by other researchers. All CC
conducted in this project utilizes wet packing method, in which the sample is
introduced to the top of the silica gel column. The CC was done to give 2
concentrated compounds coded KZY001, and KZY002.
3.6.3 Centrifugal Thin Layer Chromatography (CTLC)
3.6.3.1 Preparation of the Sorbent for CTLC
Before the use of CTLC for separation, the stationary phase has to be prepared
manually, using Silica Gel 60 PF254 Containing Gypsum as the sorbent. The
detail process is described below.
50 g of Si gel with gypsum material was introduced into a 250 ml conical flask
through the funnel. 100 ml of ice-cold distilled water (4°C) was then added
slowly into the conical flask and the content was shaken thoroughly for a good
mixing. The slurry mixture was then poured immediately into the standing
round glass plate (called rotor) with its edge fenced with cellophane tape. The
rotor was rotated gently to allow the slurry mixture to flow to every edge of
the rotor and filled the glass plate. The plate was allowed to sit in fume

9. 61
chamber for 25- 30 minutes to set before placing into an oven of 65°C
overnight for drying.
The plate was taken out for cooling the next day and processed with a
scrapping kit to be a circular thin layer of silica gel as shown at the lower left
hand corner of Figure 3.2.
The plate can then be mounted to the rotating motor instrument called
Chromatotron. The sample was introduced through an inlet tube at the centre
of Chromatotron onto the silica get plate. The rotating solvent kept in solvent
tank was then allowed to flow onto the silica get plate through a fine tubing.
The progress of the chromatography was monitored by UV light coherently
the sample would appear as several purple circular bands.
Figure 3.2: The set-up of CTLC (Harrison Research, 2014)
Rotor
Solvent tank
Chromatotron
Power supply
for rotating
motor of
Chromatotron
Chromatotron
close lid
Sample/ solvent inlet

10. 62
3.7 Characterization on Pure Compounds Obtained
3.7.1 Nuclear Magnetic Resonance Spectroscopy (NMR)
The samples were first dissolved in a minimal deuterated solvent (3 – 6 drops)
before the liquid samples being transferred to a diameter of 5 mm, borosilicate
glass NMR tube. The NMR tubes containing solutions of KZY001 (in CD3OD
δH 4.78, δC 49.15) and KZY002 (in CDCl3 δH 7.26, δC 77.23) were later topped
up to a height of 4 cm by respective deuterated solvents. TMS (δH 0.00) was
used as internal standard. The tubes were analysed by JNM ECP 400NMR
Spectrophotometer for 1
H NMR, 13
C NMR and 2-D NMR (HMQC, HMBC)
experiments.
Shimming of samples were done for each samples by setting the axis (Z1, Z2,
Z3, Z4) accordingly to obtain at least 1200 in the fine lock indicator at gain 27,
using the Delta NMR software. The multiplicity of proton is denoted as s
(singlet), d (doublet), t (triplet), dd (doublet of a doublet), td (triplet of a
doublet).
3.7.2 Infrared (IR) Absorption Spectroscopy
Samples in this research were ran as thin-film using KBr salt discs. The
dissolved concentrated samples in minimal anhydrous solvent were smeared
on one side of the discand the solvent would evaporate to leave a thin film of
sample on the surface of the salt disc before subjected for measurement by the

11. 63
Spectrum RX1 IR Spectrometer. KZY001 was analyzed using C5H5N while
KZY002 was tested using CHCl3 as solvents, respectively.
3.7.3 Mass Spectrometry (MS)
HREIMS with electron energy set to 175 eV in positive mode, was used to
confirm the molecular formula of the extracted pure compounds in this project.
The samples were dissolved in LCMS grade methanol and filtered before
injected directly into the probe of Accurate-Mass Q-TOF LC/MS model
G6520B for mass analysis.
3.7.4 Ultra Violet-Visible Light Spectrometry (UV-Vis)
Quartz cuvette was used to contain the analyte and a baseline correction (of
solvents used) was conducted in HALO SB-10 UV-VIS Single Beam
Spectrophotometer before any sample analysis. KZY001 was dissolved in
C5H5N while KZY002 was dissolved in CHCl3 for the analysis. The λmax and
the absorbance of the samples were recorded which was then used to calculate
the molar absorptivity, 𝜀 with unit L/mol.cm-1
.
3.7.5 Polarimetry
Similar to the procedures in preparing analyte for the UV-Vis experiment,
KZY001 was first dissolved in C5H5N while KZY002 was dissolved in CHCl3
before being introduced to the 10 ml polarimeter container. The container was

12. 64
then inserted in P-2000 Polarimeter for analysis. The temperature was set to
26 °C and the instrument was set to zero each time when there is a change of
analytes. The specific rotation [α], in degree unit ( °) were recorded for each
samples.

13. 65
CHAPTER 4
RESULTS AND DISCUSSION
4.1 Overview
In this chapter, the isolation of 2 pure compounds from the extract of
Andrographis paniculata by means of chromatographic methods such as CC
and CTLC were reported with the specific solvent composition in each solvent
system used. The structural elucidation of the 2 pure compounds, coded
KZY001 and KZY002 were done through systematic spectroscopic approach,
including 2D-NMR analysis. The compounds obtained were later compared
with literature data to further confirm the proposed structures.
From the characterization, KZY001 and KZY002 were identified as
andrographolide and 14-deoxy-11,12-didehydroandrographolide. The
explanations on the structural elucidation of each of the compounds were
provided in this chapter.

14. 66
4.2 Isolation of Compounds from Crude Extract
4.2.1 Filtration
In the partition extraction process described earlier (refer to Chapter 3, section
3.5), the aqueous layers from the various organic solvents were collected and
combined. Upon mixing the aqueous layers obtained, some browngranular
solids were observed at the bottom of the containers. The brown solids were
filtered and dried while the filtrates were concentrated and kept frozen for
future studies. The collected solids were dissolved in excess MeOH (due to its
low solubility) and the concentration with further drying of the solvent gave
yellowish crystals (plates) weighing 1.20 g. The crystals were the first pure
compound isolated from the plant and were coded as KZY001.
4.2.2 Column Chromatography (CC)
Dissolved CHCl3 crude (using CHCl3) was first subjected to CC, using
activated Si gel (600 g) that was manually packed with constant tapping, in an
i.d. 30 mm wide, 500 mm long glass column. Sufficient amount of cotton
wool was placed inside the glass column, on top of the stopcock in advanced,
to prevent leakage of Si gel. The eluting tip of the column was also plugged
with cotton bud to prevent introduction of Si gel into the collected fractions.
Elution of 8 hours was done using 100% CHCl3, by the flow rate of 2 drops
per second, allowing 33 fractions to be collected with TLC monitoring.

15. 67
Fr. 26 – 33, under the conducted TLC analysis, showed strong UV active spots
(strong purple) under λ 254 nm. It was later concentrated in vacuo and pump-
dried to yield clean weight of 1.28 g. Before further separation by CC, 250 g
of Si gel was packed into the same column previously mentioned with similar
procedures, and elution was done using CHCl3: MeOH (97: 3 v/ v) as the
solvent system. With the flow rate of 2 drops per second, the experiment was
done through TLC monitoring, resulting in the collection of 3 eluted fractions:
Fr. A, Fr. B, and Fr. C.
TLC analysis again showed Fr. B (0.96 g) contains UV active compounds
under short λ and was subjected to another CC, this time, in an i.d. 20 mm,
500 mm long glass column with sintered disc installed. 3 pinches of Na2SO4
salts were introduced on top of the sintered disc beforehand to prevent Si gel
leakage. 150 g of Si gel was used for packing and the elution with 3 drops per
second, was done in 12 hours using CHCl3: MeOH (97: 3 v/ v) to afford 6
fractions, B1 – B6.
4.2.3 Centrifugal Thin Layer Chromatography (CTLC)
Fr. B3 collected from CC was concentrated and subjected for further
purification using CTLC under the solvent system CHCl3: MeOH (96: 4 v/ v).
The flow rate of solvent directed from the solvent tank was adjusted to 4 drops
per second and the elution was done with visualization aid using UV lamp
under λ 254 nm. Broad UV active (strong purple) band was collected from the
chromatotron (as seen in Figure 3.4) to yield the second compound of interest

16. 68
coded KZY002. Figure 3.5 summarizes the conditioning for separation of the
pure compounds.
Figure 4.1: The circular bands were separated from each other in the Si gel,
which each of them can be collected when they move the end edge of the rotor
via centrifugal forces during the separation via CTLC.
1st
band
Base band
Under UV light, λ= 254 nm

17. 69
Figure 4.2: The isolation and separation of compounds from the aqueous layer and CHCl3 crude, after the partition extraction on the MeOH
extract of Andrographis paniculata.
From partition extraction using various solvents
Combined aqeous layer with brown solid (27.87 g)
sedimented at the bottom of the flask
Aqueous layer was kept
frozen for further studies
Subject to filtration and the filtered solids dissolved
in excess MeOH
Supernatant was collected and concentrated in
vacuo using rotary evaporator to yield yellow
plate crystals, KZY001 (1.20 g)
CHCl3 crude (49.66 g) was subjected to CC using 100%
CHCl3 to afford 33 fractions
Fr. 26 - 33 (1.28 g) subjected to CC using CHCl3: MeOH (97: 3
v/ v) to give Fr. A, Fr. B, and Fr. C
Fr. B subjected to CC again using CHCl3: MeOH (97: 3 v/ v) to
afford 6 fractions
Fr. B3 from the 6 fractions was purified by CTLC
using CHCl3: MeOH (96: 4 v/ v) to yield KZY002
(60 mg)

18. 70
4.3 Characterization and Structural Elucidation of Compounds
In this section, discussion on the characterization and the structural elucidation
were done on the 2 compounds obtained from the isolation process, which are
coded as KZY001 and KZY002. The characterizations of the natural products
were carried out using spectroscopic methods which mainly consist of NMR,
IR, MS, UV-Vis, Polarimetry and melting point experiments. The proposed
structures were later confirmed and identified by the literature data.
4.3.1 Characterization of Compound 1: KZY001
KZY001 was obtained as yellowish crystal plates (1.20 g, MeOH) with m.p.
230 – 232°C (Reported: 230 – 239°C), [α]D -123° (Reported: -126.6°), UV
λmax (C5H5N), (log ε): 261 nm, (4). The IR spectrum of the compound (Figure
4.1), obtained from KBr thin-film in C5H5N showed the presence of hydroxyl
group at 3372 cm-1
, an ester C=O with conjugation (5 membered α,β-
unsaturated-γ-lactone function) at 1752 cm-1
and 1673 cm-1
, C-O stretch
(primary alcohol) at 1068 cm-1
, C-O stretch (secondary alcohol) at 1088 cm-1
,
and exo-methylene at 880 cm-1
. The IR data are tabulated in Table 4.3.
The HR-TOF-LCMS gave a pseudomolecular ion, [M+Na]+
, at m/z 373.1988
(calculated: 373.1990), which corresponded to the molecular formula
C20H30O5. 13
C NMR spectrum with decoupling showed 20 signal peaks which
agreed to the data provided by the HR-TOF-LCMS and indirectly suggested
the natural product to be a diterpenoid type.

19. 71
Figure 4.3: IR spectrum of KZY001 obtained from KBr thin-film (C5H5N).
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0
32.1
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105.8
cm-1
%T
3854 3751
3372
3039
3008
2971
2937
2220
1928
1752
1673
1645
1613
1589
1485
1440
1379
1215
1188
1150
1088
1068
1032
998
880
750
612
574
406

20. 72
Table 4.1: The IR assignments for KZY001
Origin Wavenumber, cm-1
Assignments
O-H stretch 3372 cm-1
Hydroxyl group
Conjugated ester C=O 1752 cm-1
, 1673 cm-1
5 membered α,β-
unsaturated-γ-lactone
C-O stretch, 1068 cm-1
Primary alcohol
C-O stretch, 1088 cm-1
Secondary alcohol
C-H 880 cm-1
exo-methylene
DEPT experiment showed that KZY001 consists of a total of 2 methyl (CH3),
8 methylene (CH2), 5methine (CH), and 5 quaternary carbon (C) signals,
which gave 20 carbons and 27 hydrogen counts. The 3 missing hydrogen was
associated to the presence of 3 hydroxyl groups in the compound as the
observed hydrogen were each attached to a heteroatom (in this case, oxygen
atom), which will not show any signal in 13
C NMR spectrum.
1
H NMR spectrumof KZY001 showed two methyl signals at δH 0.75 (3H, s)
and 1.21 (3H, s), allylic function at δH 2.03 (2H, td, J = 4.80, 12.0),a presence
of ester at δH 4.16 (1H, dd, J = 2.11, 10), exo-methylene proton signals at δH
4.67 (1H, br s ) and 4.88 (1H, br s), presence of vinylic function at δH 6.84 (1H,
td, J = 1.66, 6.80).
In the down field region of the 13
C NMR spectrum, a peak at δC172.8
suggested the presence of an ester C=O which partially agrees to the data from
IR for the presence of a lactone. The olefinic region of the spectrum (along

21. 73
δC100.0 – 160.0) showed 4 peaks, indicating 4 unsaturated double bonded
carbons which can be identified under DEPT, where the 2 of the peaks (at δC
149.5, 129.9) were attributed to 2 unsaturated quaternary carbon (C=C). The
remaining 2 peaks represented the methylene of a terminal alkene (C=CH2), at
δC 109.3 and an unsaturated methine (C=CH) at δC 148.9. The signal at δ109.3
(unsaturated methylene, C=CH2) can be associated to the previously
mentioned exo-methylene from the IR (Table 4.1) as it is the only unsaturated
double bonded methylene in the 13
C NMR spectrum.
Figure 4.4: The structure of a 5 membered α,β-unsaturated-γ-lactone, in
which the oxy-methylene signal at δC 76.3 in 13
C NMR spectrum was
associated.
Along the δC 60.0 – 90.0 region, DEPT experiment showed 2 methine and 2
methylene signals in the 90° and 135° spectrum. The obvious oxy-methine
(CH-O) at δC81.0 was spotted, followed by an oxy-methylene (CH2-O) at
δC76.3, which was slightly displaced as oxy-methylene is normally observed
lying along δC 60.0 –δ 65.0. It is most probably being part of a ring system.
Hence, it can be concluded that the oxy-methylene at δC 76.3is a part of the

22. 74
cyclic ester as shown in Figure 4.4. The other 2 signal peaks at δC 66.7, 65.1
in that mentioned region were assigned as oxy-methine (CH-O) and oxy-
methylene (CH2-O) as the observed molecular formula from the mass
spectrometer did not show any other elements other than O.
In the aliphatic region (δC 20.0 – 60.0), a total of 11 peaks could be observed
from the spectrum of 13
C NMR. DEPT experiment showed 2 quanternary
carbon signals (δC 43.8, 40.1), 2 methines signals (δC 57.5, 56.4), 5 methylenes
signals (δC 39.1, 38.2, 29.1, 25.8, 25.3), and 2 methyl signals (δC 23.5, 15.6)
which did not provided any extra information about the structure of KZY001.
However, it was observed that the methyl signal, at δC 23.5 has been displaced
from its usual region (δC 10.0 – 20.0).
The proton signals from the 1
H NMR spectrum at δH 0.75 and 1.21 were
assigned to the two methyl signals at δC 23.5 (C-18) and at δC 15.6 (C-20)
respectively, which showed correlation to the signals at δC 40.1 and 43.8
(methine carbon signal, C-9 and quarternary carbon signal, C-4 respectively),
δC 56.4 and 57.5 (methine signal, C-5 and C-7 respectively), δC 65.1 (oxy-
methylene signal, C-14) and δC 80.1 (oxy-methine signal, C-3) in the spectrum
of HMBC to give a partial structure as shown in Figure 4.5.

23. 75
Figure 4.5: Partial structure constructed from the correlations that were
observed in HMBC spectrum by the protons of the two tertiary methyl C-18
and C-20.
The two br s of the exo-methylene at δH 4.67 and 4.88 in 1H NMR spectrum
were assigned to the exo-methylene (δC 109.3, C-17) from HMQC experiment
and was found to have correlation to methine signal (C-9) at δC 57.5 and
methylene signal (C-7) at δC 39.1 in the HMBC spectrum. This further
confirms the position of the methine signal (C-9) and shows a refined partial
structure in Figure 4.6below.
Figure 4.6: Refined partial structure of KZY001 after observed correlations of
the exo-methylene at C-17.

24. 76
The allylic proton signal at δH 2.03 that was connected to C-7 showed a td
splitting and correlates to another aliphatic methylene signal (C-6) at δC 25.3
in the HMBC spectrum. The multiplet splitting of a proton (δH 1.32) that was
connected to C-5 showed correlations towards C-18, C-6 and a methylene
signal at δC 38.2 (C-1) a partial homocyclic ring and confirming the previous
assigned carbon positions as shown in Figure 4.7.
Figure 4.7: Partial homocyclic ring formed when correlations in HMBC
spectrum were observed made from the td splitting proton (δH 2.03) and
multiplet signal of proton (δH 1.32).
Another multiplet signal of a proton at δ 1.79 that was assigned to a methylene
signal (C-2) at δC 29.1 showed correlations to C-3, C-4 and C-10 in the
HMBC spectrum, in which a formation of a bicyclic ring structure was seen
and revealed an ent-labdane type structure (refer to Figure 2.0, page 17). The
primary and secondary alcohol groups that was observed previously in the IR
experiment was further assigned to the structure to give structure shown in
Figure 4.8.
δH 1.32

25. 77
Figure 4.8: Refined partial structure of KZY001 showed an ent-labdane type
structure after the hydroxyl groups being added into the structure.
Multiplet splitting signal of a proton at δH 2.61 was assigned to a methylene
signal (C-11) at δC 25.8 and was found to have correlation to C-9 and towards
the vinylic carbon signals (δC 148.9, C-12 and δC 129.9, C-13) in the HMBC
spectrum. This indicated the suspected position of the lactone (since it has yet
to be assigned) observed by IR experiment (Table 4. 1).
A td splitting signal of a proton (δH 6.84) assigned to the vinylic methine
signal (δC 148.9, C-12) was found correlated to the ester C=O group (δC 172.8,
C-16), upfield oxy-methine (δC 66.7, C-14) and C-9 confirmed the lactone
ring’s position relative to the rest of the structure (shown in Figure 4.9).
Position of C-14 was confirmed when a correlation was observed from a br d
splitting signal of proton (δH 5.01) to the C-16 carbonyl.

26. 78
Figure 4.9: The lactone position in KZY001.
The presence of the 5 membered ring lactone was further confirmed when a dd
splitting signal of a proton at δH 4.16 was assigned to the lactone-associated
oxy-methylene signal at δC 76.3 (C-15) (refer Figure 4.4) was found
correlated to C-14, C-16 in the HMBC spectrum. The completion of the
structural elucidation of KZY001 eventually revealed the position of the
quaternary carbon (δC 149.5) at C-8. The hydroxyl group was assigned onto C-
14 to compensate the molecular formula, C20H30O5 that was provided by the
HR-TOF-LCMS. The proposed structure of KZY001 was shown in Figure
4.10.
H

27. 79
Figure 4.10: The partial structure A was later refined to give proposed
structure B of KZY001.
KZY001 was taken to compare with the literature data (Matsuda, et al. 1983)
which eventually confirmed the structure and was identified as
andrographolide (shown in Figure 4.11), the major constituent of
Andrographis paniculata.
Figure 4.11: Strucutre of andrographolide (as compared to literature data
from Matsuda, et al., 1983).

28. 80
It was seen that andrographolide consists of an unsaturated group at C-12 and
C-13 and a nearby hydroxyl group at C-14 which suggested the compound has
undergone an epoxidation reaction. A hypothetical biosynthesis scheme was
established and shown below in Figure 4.12.
Figure 4.12: The hypothetical biosynthesis mechanism of andrographolide
(Adapted from Matsuda, et al., 1983).
During the hypothetical biosynthesis, the double bond of compound at (i)
donates electrons to electrophilic oxygen to form an epoxide (ii). Due to the
steric constraint of the epoxide, the ring is cleaved to form the hydroxyl group.
The electrons were balanced from the transition state (ii) and form a double
bond which gives the structure of andrographolide (iii).
A summary of the overall data is presented in Table 4.2. The 13
C and 1
HNMR
spectra with the proposed structure are presented in Figure 4.13 and Figure
4.14 respectively.
(i) (ii)
(iii)

29. 81
Table 4.2: Summary of spectral data for KZY001
Carbon 13
C NMR, δC *Literature
data, δC
1
H NMR, δH
1 38.2 (CH2) 37.3 -
2 29.1 (CH2) 29.0 1.79, m
3 81.0 (CH-O) 79.8 -
4 43.8 (C) 43.2 -
5 56.4 (CH) 55.3 1.32, m
6 25.3 (CH2) 24.3 -
7 39.1 (CH2) 38.1 2.03, td, J = 4.80,
12.0
8 149.5 (C=C) 147.9 -
9 57.5 (CH) 56.3 -
10 40.1 (C) 39.1 -
11 25.8 (CH2) 25.0 2.61, m
12 148.9 (CH=C) 147.0 6.84, td, J = 1.66,
6.80
13 129.9 (C=C) 130.2 -
14 66.7 (CH-O) 66.0 5.01, br d, J = 5.9
15 76.3 (CH2-O) 75.4 4.16, dd, J =2.10,
10
16 172.8 (C=O) 170.7 -
17 109.3 (CH2=C) 108.8 4.67, br s;
4.88 br s
18 23.4 (CH3) 23.7 0.75, s
19 66.7 (CH2-O) 64.1 -
20 15.6 (CH3) 15.2 1.21, s
*Literature value were taken from (Matsuda, et al., 1983)

30. 82
203 18
8
16 12
13 17
15 14 19 9 5 4 7
10
1 2
11
6
Figure 4.13: 13
C NMR spectrum for KZY001 (100 MHz, CD3OD)

31. 83
12 td
14
br d
17 br s
17’
br s
20 s
18 s
11
7
2 5
m
mm
td
Figure 4.14: 1
H NMR spectrum for KZY001 (400 MHz, CD3OD)

32. 84
4.3.2 Characterization of Compound 2: KZY002
KZY002 was isolated as white amorphous powder (60mg, CHCl3) with m.p.
202 – 204°C (Reported: 203 – 204°C), [α]D +76° (Reported: +54°), UV λmax
(CHCl3), (log ε): 251 nm, (4). The IR spectrum of the compound (Figure
4.15), obtained from KBr thin-film in CHCl3 showed the presence of hydroxyl
group at 3365 cm-1
, an ester C=O with conjugation (5 membered α,β-
unsaturated-γ-lactone function) at 1750 cm-1
and 1644 cm-1
, C-O stretch
(primary alcohol) at 1036 cm-1
, C-O stretch (secondary alcohol) at 1082 cm-1
,
and exo-methylene at 894 cm-1
, of which were similar to that of
andrographolide, the 1st
compound that was discussed earlier. The IR data are
tabulated in Table 4.3.
Table 4.3: The IR assignments for KZY002
Origin Wavenumber, cm-1
Assignments
O-H stretch 3361 cm-1
Hydroxyl group
Conjugated ester C=O 1747 cm-1
, 1644 cm-1
5 membered α,β-
unsaturated-γ-lactone
C-O stretch, 1036 cm-1
Primary alcohol
C-O stretch, 1082 cm-1
Secondary alcohol
C-H 894 cm-1
exo-methylene

33. 85
Figure 4.15: IR spectrum of KZY002 obtained from KBr thin-film (CHCl3).
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.0
52.7
54
56
58
60
62
64
66
68
70
72
74
76
78
80
82
84
86
88
90
92
94
96
98.7
cm-1
%T
3361
3079
2932
2850
1747
1644
1573
1446
1386
1347
1264
1215
1131
1082
1036
1001
954
923
894
812
756
666
633
579

34. 86
The HR-TOF-LCMS gave a pseudomolecular ion, [M+Na]+
, at m/z 355.1884
(calculated: 355.1885), which corresponded to the molecular formula
C20H28O4, suggesting seven double bond equivalents. The molecular formula
of KZY002 showed a difference of two hydrogen and an oxygen atom as
compared to andrographolide (C20H30O5). 13
C NMR spectrum of KZY002
with decoupling showed 20 peaks which agreed to the data provided by the
HR-TOF-LCMS and suggested the natural product to be a similar diterpenoid
type.
DEPT experiment showed that KZY002 consists of a total of 2 methyl (CH3),
7 methylene (CH2), 6 methine (CH), and 5 quaternary carbon (C) signals
which gave 20 carbons and 26 hydrogen counts. The 2 missing hydrogens
were associated to the 2 hydroxyl groups in the compound as the observed
hydrogen were each attached to an oxygen atom, which will not show any
signal in 13
C NMR spectrum. An addition of methine and a loss of a
methylene signals observed in the 13
C spectrum suggested an addition of an
unsaturated group in the KZY002, as compared to the structure of
andrographolide.
1
H NMR spectrum of KZY002 showed the two methyl signals at δH 0.81 (3H,
s) and 1.25 (3H, s), allylic function at δH 2.30 (1H, d, J = 2.16), exo-methylene
signal at δH 4.51 (1H, br s) and 4.78 (1H, br s ), ester function at δH 4.81 (1H,
br s), vinylic function at δH 6.15 (1H, overlapped dd, J = 15.5) and 6.88 (1H,
dd, J = 10.3, 15.8).

35. 87
Using the 1
H NMR, 13
C NMR data obtained and the observed HMBC
correlations, the bi-homocylic partial structure of KZY002 was established,
which was found to be similar to that of andrographolide, where both have an
ent-labdane type structure (Figure 4.8, page 76).
A doublet splitting signal of an allylic proton (at δH 2.30) that was assigned to
a methine signal (δC 61.8, C-9) showed correlations to the methyl signal at δC
16.0 (C-20), quaternary carbon signal (δC 38.4, C-10), exo-methylene signal
(δC 109.3, C-17), unsaturated methine signals (δC 121.2 and 136.1, referring to
C-12 and C-11 respectively), and another quaternary carbon signal (δC 148.2,
C-8) in HMBC spectrum which confirmed the previous assigned partial
structure and revealed the possible position of the olefinic group (C-11, C-12)
as shown in Figure 4.16 below.
Figure 4.16: The observed correlation at C-9 confirmed the previous assigned
structure and revealed the possible position of suspected olefinic group at C-
11 and C-12.

36. 88
A br dd splitting signal of a vinylic proton (δH 6.15) assigned to C-12 showed
correlations to C-9 and C-11 in the HMBC spectrum, which confirmed the
attachment of C-11 and C-12 and the position of the olefinic group connected
to C-9. The observed correlation also revealed the quaternary carbon signal
(δC 129.4, C-13), unsaturated methine signal (δC 143.1, C-14) and the ester
C=O signal (δC 172.5, C-16) that were all associated to the lactone (observed
by IR) as shown in Figure 4.17.
Figure 4.17: Correlations from HMBC that revealed the partial lactone
structure from the proton (δH 6.15) attached to C-12.
The structural elucidation was done when a broad singlet signal at δH 7.18 that
was assigned to C-14 showed correlation to the oxy-methylene signal (δC69.8,
C-15) in the HMBC spectrum, which completed the lactone structure. This
was further confirmed by the observed correlations of the proton of C-15 (at
δH 4.81) towards the C-11(5 bonds apart), C-12 (4 bonds apart due to
conjugation), C-13, C14, and C-16. The complete structure of KZY002 was
shown in Figure 4.18.

37. 89
Figure 4.18:The presence of the lactone was confirmed when correlations of
proton signals at δH 7.18 and 4.81 were observed form the HMBC spectrum.
KZY002 was taken to compare with the literature data (Matsuda, et al. 1983)
which eventually confirmed the structure and identified as 14-deoxy-11,12-
didehydroandrographolide (Figure 4.19), another major constituent of
Andrographis paniculata.
Figure 4.19: Structure of 14-deoxy-11,12-didehydroandrographolide
(Compared with literature data).

38. 90
As mentioned in the earlier characterization process, KZY002 was suspected
to be lack of one hydroxyl group, which proved to be true where the hydroxyl
group at C-14 in andrographolide was seen missing in 14-deoxy-11,12-
didehydro-andrographolide after the characterization. The occurring of this
compound could be due to the weak allylic hydroxyl at C-14 which is
susceptible to elimination and forms a double bond after rearrangement. A
hypothetical biosynthesis scheme of 14-deoxy-11,12-didehydro-
andrographolide was established and shown in Figure 4.20 below.
Figure 4.20: The hypothetical biosynthesis mechanism of 14-deoxy-11,12-
didehydroandrographolide (Adapted from Matsuda, et al, 1983).
The allylic alcohol was eliminated from the lactone of andrographolide and
was balanced by the donation of electrons from the double bond (i). This
(i)
(iii)
(ii)
-H+

39. 91
leaves a carbocation which was subsequently followed by a proton abstraction
that would give 14-deoxy-11,12-didehydroandrographolide (iii).
A summary of the overall data is presented in Table 4.4. The13
C and 1
HNMR
spectra with the proposed structure are presented in Figure 4.21 and Figure
4.22 respectively.

40. 92
Table 4.4: Summary of spectral data for KZY002
Carbon 13
C NMR, δC *Literature
data, δC
1
H NMR, δH
1 38.7 (CH2) 38.7 1.55, dt, J = 3.4,
13.2
2 28.2 (CH2) 28.8 1.75, overlapped
br s
3 80.9 (CH-O) 80.1 -
4 43.1 (C) 43.3 -
5 54.8 (CH) 54.7 -
6 23.1 (CH2) 23.6 -
7 36.7 (CH2) 37.0 2.53, m
8 148.2 (C=C) 149.2 -
9 61.8 (CH) 61.7 2.30, d, J = 2.16
10 38.4 (C) 39.0 -
11 136.1 (CH=C) 135.6 6.88, dd, J = 10.3,
15.8
12 121.2 (CH=C) 121.9 6.15, overlapped
dd, J = 15.5
13 129.4 (C=C) 128.8 -
14 143.1 (CH=C) 145.1 7.18, br s
15 69.8 (CH2-O) 70.3 4.81, br s
16 172.5 (C=O) 172.8 -
17 109.3 (CH2=C) 108.7 4.51, br s;
4.78 br s
18 22.8 (CH3) 23.6 1.25, s
19 64.3 (CH2-O) 64.2 4.16, m
20 16.0 (CH3) 16.0 0.81, s
*Literature value were taken from (Matsuda, et al., 1983

41. 93
16
6
18
202
8 14
11
13
12 17
3
15
19 9 5 4 1
7
10
Figure 4.21: 13
C NMR spectrum for KZY002 (100 MHz, CDCl3)

42. 94
Figure 4.22: 13
C NMR spectrum for KZY002 (400 MHz, CDCl3)
1
2
912
11
14
15
17
17’
18
20
19
dt
s
s
br s
dd
dd
br s
br s
br s
m
d
br s