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.

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

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.

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.

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.

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.

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.

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

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

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 1H NMR,

13C 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

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

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.

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.

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.

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

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

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)

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.

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

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.

1H 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

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

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 1H 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.

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.

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

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.

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

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).

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 1HNMR

spectra with the proposed structure are presented in Figure 4.13 and Figure

4.14 respectively.

(i) (ii)

(iii)

81

Table 4.2: Summary of spectral data for KZY001

Carbon 13

C NMR, δC *Literature

data, δC

1H 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)

82

20 3 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)

83

12 td 14

br d

17 br s

17’

br s

20 s

18 s

11 7

2 5

m m m

td

Figure 4.14: 1H NMR spectrum for KZY001 (400 MHz, CD3OD)

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

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

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.

1H 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).

87

Using the 1H NMR,

13C 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.

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.

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).

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+

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 1HNMR

spectra with the proposed structure are presented in Figure 4.21 and Figure

4.22 respectively.

92

Table 4.4: Summary of spectral data for KZY002

Carbon 13

C NMR, δC *Literature

data, δC

1H 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

93

16

6

18

20 2

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)

94

Figure 4.22: 13

C NMR spectrum for KZY002 (400 MHz, CDCl3)

1

2

9 12

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