Nama: Hayatun Nisa, Sisna, Siti Hawa

Nama: Fuad amrillah,Rini Maulida, Listy Amalia Annisa Putri

Instrumental equipment. A Waters Associates liquid chromatograph (Milford, MA,

USA), equipped with isocratic 6000 A pump, U6 K injector, [[(amino propyl) methyl]
silyl]-bonded amorphous silica column (μBondapack/carbohydrate analysis, 39 mm
300 mm, particle size 10 μm), differential refractometer R401, and Waters Data
Module 745 integrator was used. The mobile phase was acetonitrile:water (80:20).
Operating conditions were flow rate of 0.9 ml/min and ambient temperature.

Nama: Arief Ersyandi, Fitri Norhasanah, Syarifah Alawiyah

Summary
A simple and rapid method for analysis of four major triterpenoids in Centella asiatica
preparations is described. Thin-layer chromatography is widely used for saponin
analysis; here it was used for identification and quantification of the pure compounds
and the compounds in complex mixtures, by densitometric analysis after TLC.
Ethanolic extracts of leaves from C. asiatica and authentic standards of asiatic acid,
asiaticoside, madecassic acid, and madecassoside, were separated by normal-phase
TLC with chloroform–methanol–acetic acid–water 60:32:12:8 (v/v) as mobile phase.
These conditions enabled successful separation of the triterpenoid acids and their
respective glycosides from other components of the crude extracts. The separated
compounds were detected with anisaldehyde–sulfuric acid solution. The intensity of
the resulting violet spots was proportional to the amount of saponin or sapogenin
present. The correlation coefficients of calibration plots were between 0.9904 and
0.9982 and the plots were linear over the range 1.25–10 nmol standard, corresponding
to approximately 0.6–5 μg for the acids and 1.2–10 μg for the glycosides. When the
method was used for analysis of the specific saponins in C. asiatica leaves the results
were consistent with literature reports.

Nama: Erwin abdillah,Delima rezka yanti,Seftiani rahmah

2.6.2. UPLC-DAD–ESI-MS analysis

Samples were analyzed by UPLC-DAD–QQQMS using an Agilent ZORBAX SB-
C18 RRHT column (50 􏰁 2.1 mm i.d.; particle size 1.8 lm) at 30 °C. The mobile
phase consisted of 0.1% formic acid in H2O (solvent A) and 0.1% formic acid in
acetonitrile (solvent B). The elution program was set up as: 0 min, 0% B; 0.4 min,
13% B; 1min, 13% B; 2min, 21% B; 3min, 31% B; 3.5min, 40% B; 4 min, 48% B;
4.01–5.5 min, 100% B. Post time: 1.5 min. The injection volume was 2 lL and the
flow rate was kept at 0.4 ml/min. MS2 scan mode (negative) was used for qualitative
analysis (Fig. 2). For quantification of the components, multiple reaction monitoring
(MRM) or selective ion monitoring (SIM) mode was used with positive polarity for
compounds 7–11 and negative polarity for compounds 1–6. Compound 1 was
analyzed at MRM 191 and 85, compound 2 at MRM 353 and 191, and compound 8 at
MRM 330 and 312 after in-source fragmentation. Other com- pounds were quantified
using SIM mode. The ion source parame- ters were set as following: capillary, 4kV;
gas temperature, 350 °C; gas flow, 11 L/min; nebulizer, 45 psi

Nama: Wira norprasetya, muthia narita, fauziah

Compound 1 appeared at (tR=3.406 min) and displayed a protonated molecule at m/z
163.1231 [M+H]+ with molecular formula C10H14N2, DBE=5 supporting one cyclic
ring and a pyridine nucleus. MS/MS fragmentation (Figure 2) showed peaks at m/z
134.0987 [M +H- 29.0244]+ empathized loss of N-CH3 group, m/z 120.0814
[M+HNC2H5]+, m/z 106.0662 [M+H-C3H7N]+ and m/z 79.0556 for pyridinium
cation [C5H4N]+ which confirmed the MS fragmentation pattern of nicotine [16]
previously isolated from Nicotiana tabaccum L. [17] and this is the first report in the
genus Caryota.

Compound 2 eluted at (tR=15.117 min) and displayed a protonated molecule at m/z

158.1168 [M+H]+ with molecular formula C8H15NO2, DBE=2 supporting one cyclic
ring and a carbonyl group. MS/MSfragmentation (Figure 2) showed peaks at m/z
143.0702 [M+H-CH3]+, m/z 99.1027 [M+H-59.0141]+ corresponding to loss of
methyl carboxylate group and this confirmed the MS fragmentation pattern of methyl
N-methyl piperidine-3-carboxylate previously detected in Areca catechu L. [5] and
this is the first report in the genus Caryota.

Compound 3 with (tR=15.585 min) appeared as a protonated molecule at m/z

186.1484 [M+H]+ with molecular formula C10H19NO2, DBE=2 expressed by
presence of one cyclic ring and a carbonyl group. MS/MS fragmentation (Figure 2)
showed peaks at m/z 171.0732 [M+H-CH3]+, m/z 157.0836 [M+H-C2H5]+, m/z
143.0725 [M+H-C3H7]+, m/z 99.1020 [M+H-C4H7O2]+ explaining loss of propyl
carboxylate group. This MS fragmentation pattern helped us to identify the compound
as propyl N-methyl piperidine-3-carboxylate
suggesting it to be a new compound that is firstly isolated from the genus Caryota.

Compound 4 appeared at (tR=15.662 min) exhibited a protonated molecule at m/z

172.1326 [M+H]+ with molecular formula C9H17NO2, DBE=2 confirmed the
presence of one cyclic ring and a carbonyl group. MS/MS fragmentation (Figure 3)
showed peaks at m/z 157.0828
[M+H-15.0498]+ with loss of methyl group, m/z 143.0715 [M+HC2H5]+, m/z
99.1018 [M+H-C3H5O2]+ corresponding to loss of ethyl carboxylate group and
matched with MS fragmentation pattern of ethyl N-methyl piperidine-3-carboxylate
previously detected in Areca catechu L. [5] and this is the first report in the genus
Caryota .

Compound 5: (tR=15.749 min) resulted in a protonated molecule at m/z 142.0858

[M+H]+ with molecular formula C7H11NO2, DBE=3, which mean that one
monounsaturated cyclic ring and a carbonyl group are present. MS/MS fragmentation
(Figure 3) exhibited peaks at
m/z 127.0745 [M+H-CH3]+, m/z 83.0502 [M+H-59.0356]+ with loss of methyl
carboxylate group which is similar to MS fragmentation of guvacoline previously
identified in Areca catechu L. [5] and this is the first report in the genus Caryota.

Compound 6: eluted at (tR=15.850 min) showed a protonated molecule at m/z

170.1163 [M+H]+ with molecular formula C9H15NO2, DBE=3 supporting the
presence of one monounsaturated cyclic ring and a carbonyl group. MS/MS
fragmentation (Figure 3) showed peaks at m/z 141.1009 [M+H-29.0154]+ with loss of
ethyl group, m/z 97.0679 [M+H-73.0484]+ with loss of ethyl carboxylate group, m/z
82.0654 [M +H-C4H8O2]+ that confirm MS fragmentation of Ethyl N-methyl-l,
2,5,6-tetrahydro-pyridine-3-carboxylate previously identified in Areca catechu L.[5]
and this is the first report in the genus Caryota.

Compound 7: (tR=17.376 min) displayed a protonated molecule a m/z 156.1016

[M+H]+ with molecular formula C8H13NO2, DBE=3 supporting the presence of one
monounsaturated cyclic ring and a carbonyl group. MS/MS fragmentation (Figure 4)
showed peaks at m/z 141.0917 [M+H- CH3]+, m/z 97.0664 [M+H-59.0352]+ with
loss of methyl carboxylate group, m/z 82.0663 [M+H-C3H6O2]+ which is similar to
MS fragmentation of arecoline previously detected in Areca catechu L. [5] and this is
the first report in the genus Caryota.

Compound 8: (tR=21.353 min) displayed a protonated molecule at m/z 152.0681

[M+H]+ with molecular formula C8H9NO2, DBE=5 confirming one cyclic ring and a
pyridine nucleus. MS/MS fragmentation (Figure 4) showed peaks at m/z 123.0486 [M
+H-29.0195]+ with loss of ethyl group, m/z 79.0542 [M+H-73.0139]+ with loss of
ethyl carboxylate group, which was in accordance with the MS fragmentation pattern
of ethyl nicotinate previously identified in Areca catechu L. [5] and this is the first
report in the genus Caryota.

Nama: Hj. Lailatanor, akbarina anwar, keke indriani

Chromatographic analysis were carried out using an HPLC system consisted of

two Shimadzu LC-10ATvp chromatographic pumps (Kyoto), a Develosil Combi RP-5
C30-column (50 x 4.6 mm, i.d., 5 μm, Nomura Chemical, Tokyo), and a Shimadzu
SPD-10 AV UV-VIS detector (Shimadzu), and a 7125 injector with a 20 μl sample
loop (Rheodyne, CA, U.S.A.).
Mobile system consisting of (A) a mixture of acetonitrile and water (80:20,v/v)
containing 0.05% TEA and (B) a mixture of acetonitrile, methanol and ethyl acetate
(68:5:27, v/v/v) containing 0.05% TEA. The gradient was programmed as follows: 0-
4 min, 1-10% B; 4-25 min, 50-80% B; 25-35 min, 100% B; and 35-45 min, 1% B.
The separated carotenoids were detected and measured at 450 nm. The total
chromatographic run time was 45 min at flow rate of 1 ml/min.

Nama: Mita Kurniati, Noor Redha Rezky dan Syafinah Wahdah

2.2 Thin-layer chromatography (TLC)

The pigment composition of green tea crude extract was analyzed with silica gel
GF254 as stationary phase and hexane : diethyl ether : acetone (6 : 3 : 2, v/v/v) as
mobile phase. The colour of each spot on TLC plate was observed and the Rf value
was calculated.

Nama: Pahriah ,Muhammad Faisal Firdaus

HPLC-DAD system for analysis of phenolic compounds. Chromatographic separation

was achieved as previously reported (Amaral et al., 2004) with an analytical HPLC
unit (Gilson), using a reversed-phase Spherisorb ODS2 (250_4.6 mm, 5 lm particle
size, Merck, Darmstadt, Germany) column. The solvent system used was a gradient of
water/formic acid (19:1) (A) and methanol (B), starting with 5% methanol and
installing a gradient to obtain 15% B at 3 min, 20% B at 5 min, 25% B at 12 min, 30%
B at 15 min, 40% B at 20 min, 45% B at 30 min, 50% B at 40 min, 70% B 45 min and
0% B at 46 min. The flow rate was 1 mL min1, and the injection volume was 20 lL.
Detection was accomplished with a diode array detector (DAD) (Gilson), and
chromatograms were recorded at 320 and 350 nm. Spectral data from all peaks were
accumulated in the 200–400 nm range. Data were processed on an Unipoint system
software (Gilson Medical Electronics, Villiers le Bel, France).

Nama: Aden supratman oleo w, agustina, rayan

Nama: Noor Sabella Alfitri ,Nanda Arnia Sarly ,Aulivia Febrian

Thin layer chromatography study for phytochemical profiling

Each sample solution and standard were spotted by automatic sampler (Automatic
TLC Sampler 4, CAMAG) on a 60 F254 silica gel plate (Merck, Germany) and
subsequently eluted using solvent system of toluene, ethyl acetate, and formic acid
in ratio of 58: 33: 9 (v/v/v) with track distance 0.50 cm and the migration distance
was 90 mm. Detection was done under ultraviolet at 254 and 366 nm without
chemical treatment. In addition, all spots clearly visible at white light and 366 nm
after derivatized with alumunium chloride (AlCl3) 1% (%w/v in ethanol) and
sulphuric acid (H2SO4) 10% (%v/v in water). CAMAG TLC Visualizer with
winCATS software was used for imaging, analysis, and documentation of TLC.
Nama: Renaldi Satria Darmawan, Wenty safitri