Isolation and Characterization of Brassicasterol from Matoa’s N-Hexane Fraction

(Pometia pinnata) and its Toxicity Test

 
Yoan De Nanda Herru1, Adlis Santoni1, Mai Efdi1*
1 
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Andalas
University
Phone: + 62-751-71671, Fax: + 62-751 -73118 , Post: 25163
* 
e-mail: [email protected]
 
DOI:

ABSTRACT
Pometia pinnata leaves were extracted and fractionated using n-hexane,
dichloromethane, ethyl acetate, and methanol. The four fractions obtained were
screened for cytotoxic testing using the Brine shrimp lethality test (BSLT) method, n-
hexane fraction has the highest LC50 419,855 mg/L. The n-hexane fraction was
continued for the isolation stage and a secondary metabolite compound was obtained,
namely brassicasterol. The structure of this secondary metabolites was determined using
spectroscopic methods (UV-Vis, FTIR, and NMR).

Keywords: brassicasterol, brine shrimp lethality test

(BSLT), characterization, cytotoxic,
Pometia pinnata.

Introduction
Matoa or Kasai (Pometia pinnata) is a (Garuda & Syafruddin, 2014) and also
tropical plant belonging to the there are used as dye batik cloth
Sapindaceae family that has spread (Haerudin & Farida, 2017). This plant
throughout the tropics, especially in has been carried out various kinds of
Indonesia (Martiningsih, Widana, & bioactivity tests. Some of which have
Kristiyanti, 2016). In traditional plants been reported by the researchers before,
used by the community as a remedy like antioxidant (Martiningsih et al.,
injury (Lely, 2016), fever, and fatigue, 2016), anti-microbial (Lely, 2016), the
as well as anti-infection of wounds treatment of anti-HIV-1 (Suedee,
Tewtrakul, & Panichayupakaranant, rhamnoside, quercetin-3-O-rhamnoside,
2013) antihyperglycemic (Paris and proanthocyanidin A. Other
Mataputun, Rorong, & Pontoh, 2013) secondary metabolite compounds
(Paris Mataputun et al., 2013) and reported in the leaves are the glycolipid
cytotoxic (Trimedona, Nurdin, Darwis, group in the form of 1-O- palmitoyl-3-
& Efdi, 2018). For the cytotoxic test, O- [α-D-galactopyranosyl- (1 → 6) -β-
the air is based on research conducted D-galactopyranosyl] -sn-glycerol, a
by (Trimedona et al., 2018) in the know steroid glycoside group in the form of
that the fraction with n use-values LC stigmasterol-3-O-glucoside and a
50 relative contained in the n-hexane pentacyclic triterpenoid group, namely
fraction Pometia pinnata with LC50 3-O- α-L arabinofuranosyl (1 → 3) - [α-
33.0958 ± 4.5722 mg / mL, L-rhamnopyranosyl (1 → 2)] - α-L-
This plant‘s secondary metabolites arabinopyranosyl - hederagenin (Suedee
have been isolated by (Mohammad, et al., 2013). Based on the research
Noorwala, Ahmad, Zahoor, & Lajis, report above, it is known that isolation
(2012) was new triterpenoid saponin is still rarely carried out using the n-
group, namely pometin, kaemferol 3-O- hexane Pometia pinnata fraction.
α-L-rhamnopyranosid, and 3-O- [α-L- Materials and method
arabinofuranosil- (1 → 4) -α-L- Chemical and reagents
rhamnopyranosyl- (1 → 2) -α- L The materials used include dry Pometia
-arabinopyranosyl] -hederagenin pinnata leaves, organic solvents such as
isolated with methanol extract on the n-hexane, dichloromethane, chloroform,
leaves and bark of Pometia pinnata. ethyl acetate and methanol, distilled
The leaves were isolated secondary water, aluminum foil, cotton, silica gel
metabolites which belong to the other 60 (0.063-0,200 mm / Merck), and
triterpenoid saponins in the form of 3- chromatography plate. Merck's DC-
O- [α-L-arabinofuranosyl- (1-4) - α-L- AlufolienKieselgel 60 F254 thin layer
rhamnopyranosyl- (1-2) - α -L- (20x20 cm). The reagents used to test
arabinopyranosyl] - hederagenin. the content of secondary metabolites are
(Mohammad et al., 2012) and the Iron (III) Chloride, Mercury (II)
flavonoid group in the form of Chloride, Potassium Iodide,
epicatechin, kaempferol-3-O- Concentrated Sulfuric Acid, Acetic
Anhydride, Sodium Hydroxide, Lantah, Montolalu, & Reo (2017) and
Magnesium Powder, and Ammonia. (Alegantina & Isnawati, 2010)
The appearance of stains on thin-layer Activity Test of Cytotoxic from
chromatography was used 10% H2SO4 Pometia pinnata fraction using Brine
and Liebermann-Buchard reagent. Shrimp Lethality Test (BSLT)
Materials for the cytotoxic activity test Method
are shrimp larvae, seawater, Dimethyl Screening of cytotoxic activity from
Sulfoxide (DMSO). fraction was carried out according to the
method described by (Musa, 2012).and
Extraction and Fractionation
(Al-Saeedi, Al-Ghafri, & Hossain,
Pometia pinnata leaves sample were
2017). For LC50 determination was
obtained from the Andalas University
calculated using the following formula
Campus area, Limau Manis, Padang.
by (Gelani & Uy, 2016)
Pometia pinnata leaves (13 kg) were
Isolation and Purification
cleaned and air dried, then mashed
The n-hexane fraction (30 grams) was
using a grinder and obtained 6 kg of dry
separated using a chromatography
sample.
column with a silica gel stationary
6 kg of dry powder of Pometia pinnata
phase (0.063-0.200 mesh) and the
leaves was macerated using 5 L of
mobile phase in the form of n-hexane:
methanol as a solvent with 6 repetitions.
ethyl acetate (10: 0 - 0:10) and ethyl
The extract obtained was then
acetate: methanol (10 : 0 - 9,5: 0,5).
concentrated using a rotary evaporator
Eluate is stored in the vial, then grouped
to obtain a concentrated extract of 180 g
according to the separation pattern, and
of methanol. The concentrated methanol
obtained 17 subfractions (A-Q).
extract was fractionated successively
Subfraction is re-monitored the
using n-hexane, dichloromethane, and
separation pattern by the thin-layer
ethyl acetate.
chromatography method. fraction G is
Phytochemical Screening
selected to proceed to the purification
Secondary metabolites in Pometia
stage. The impurity fraction G was
pinnata as terpenoid, steroid, phenols,
dissolved using n-hexane and ethyl
alkaloid and coumarin were investigated
acetate until a white, needle-shaped
with the standard method described by
crystal formed. This white solid was
continued for purity test using the thin- steroids, flavonoids, and phenolics.
layer chromatography method and Other studies have also reported the
melting point test. results of phytochemical screening from
RESULTS AND DISCUSSION the n-hexane extract of Pometia pinnata
Phytochemical Screening leaves containing alkaloid, flavonoid,
Pometia pinnata’s extract was tested by terpenoid, and tannin class compounds
phytochemical to determine the class of (Kuspradini, Pasedan, & Kusuma,
compounds present in the extract by 2016). The hypothesis is that the
testing the alkaloids, flavonoids, dominant compound in Pometia
phenolics, terpenoids-steroids, pinnata leaves is in the form of
coumarin, and saponins. Based on the phytosterols because of the results of
results of the phytochemical test, the research from other researchers which
positive extract contained flavonoids, states that phytosterols are the most
phenolics, and terpenoids-steroids. This dominant compounds in seed plant
is following the research that has been isolation including open seeds and
done (Trimedona, Nurdin, Darwis, & closed seeds (Tonius, Wibowo, &
Efdi, 2015), that the Pometia pinnata Idiawati, 2016).
plant extract contains triterpenoids,

N - Hexane Fraction Dichloromethane Fraction

8
8
6
6
Probit Value

y = 5,245x - 9,780
Probit Value

y = 4,681x - 7,278 4
4
2
2
0
0
-2
-2 1.5 2.0 2.5 3.0 3.5
1.5 2.0 2.5 3.0 3.5 Log Concentration
Log Concentration
 Ethyl Acetate Fraction Methanol Fraction
8 6

6
y= 5,262x - 9,874 y = 4,976x - 9,243
Probit Value

Probit Value
4

2 2

0
0
-2
1.5 2.0 2.5 3.0 3.5
-2
Log Concentration
1.5 2.0 2.5 3.0 3.5
Log Concentration

Figure 1. Brine Shrimp Lethality Test result

Cytotoxic Screening mg / L and methanol of 727.78 mg. This

Brine Shrimp Lethality Test used in this is following previous research
research to determine which fraction conducted by Trimedona et al., 2018 in
has the highest LC50. Determine LC50 a cytotoxic test on the bark of Pometia
use the regression equation, it can be pinnata that n-hexane extract is more
seen below: toxic than ethyl acetate, acetone, and
Regression equation: methanol extracts. The toxicity
Y = a + bX possessed by the n-hexane fraction in
Information: the leaves of Pometia pinnata is
Y: Probit value, percentage of deaths classified as moderately toxic. A
X: Logarithm of concentration of test compound is known to be highly toxic
material if the LC50 value of lettuce is in the
a: Constants range 0-100 µg / mL, moderate toxicity
b: Slope / slope (Gelani & Uy, 2016) is at 100-500 µg / mL, low toxicity is at
Based on the test results shows that the 500-1000 µg / mL and is not toxic
n-hexane fraction has strong toxicity above 1000 µg / mL (Gelani & Uy,
properties compared to 2016). N-hexane fraction selected for
dichloromethane, ethyl acetate, and isolation and purification step.
methanol fractions. With the LC50 Isolation of Secondary Metabolites
values in sequence, namely n-hexane of The compounds isolated in this research
419,855 mg / L, dichloromethane of are sterol group compounds. This has
647.99 mg / L, ethyl acetate of 671.06 been proved with a purity test using the
thin layer chromatography method with of a double bond between the carbons
the help of spot visors in the form of (C=C), but this double bond does not
Liebermann-Buchard reagent and 10% conjugate. This happens because of the
H2SO4. Testing with thin layer electron transition from π to π *. This is
chromatography method produces a also confirmed by the thin-layer
single red-purple stain on the thin layer chromatography test, where the
chromatography plate which indicates chromatography plate that has been
that the isolated compound is a sterol spotted with eluent with UV light at a
group compound (Gerlach, Gadea, wavelength of 356 nm does not glow.
Silveira, Clerc, & Lohézic-le, 2018). So this proves that pure compounds
The second method to determine if the have C = C bonds that are not
isolated compound is pure is to use the conjugated (Abubakar, Achmadi, &
melting point test method. The results Suparto, 2017).
of the melting point test show that the IR spectroscopic measurements
isolated compound has a melting point were carried out to determine the
range of 154-155 oC. This shows that functional group structure of the
the isolated compound has been pure isolated steroid group compounds.
where the melting point range of a pure There is absorption at wave number
compound is 1oC. From these two purity 3416.67 cm-1 which indicates that there
test data, there are secondary is a hydroxyl group (-OH) which is
metabolites of the sterol group which is indicated by the presence of strain
similar to the data above. The between oxygen and hydrogen. This is
compound that have been isolated are also evidenced in the fingerprint area,
predicted as a brassicasterol compound. namely the absorption at the wave
This analysis is also strengthened by number 1023.38 cm-1, which indicates
UV, IR, and NMR spectroscopic that there are C-O groups experiencing
analysis. vibrations. The absorption at
Spectroscopic Analysis wavenumbers 2933.04 cm-1 and 2862.40
This isolated compound had maximum cm-1 indicates the presence of primary
absorption at a wavelength of 245nm C-H groups and also secondary C-H
from UV spectrum analysis. The groups which are aliphatic. This is
absorption value indicates the presence evidenced by the absorption at wave
 number 1456.88 cm-1 which is the NMR and 13
C-NMR isolated steroid
fingerprint area for the C-H group of compounds were compared with
alkanes. chemical shift data from the literature
The absorption at wavenumber (Jinming, Lin, & Jikai, 2001). Based on
1639.59 cm-1 indicates that the isolated comparative literature data, it is known
steroid compound has a non-conjugated that the predicted isolated steroid
C=C double bond. This is evidenced by compounds are brassicasterol
the absence of wave number absorption compounds. Brassicasterol is a sterol
in the 1500 cm-1 fingerprint area. Based compound (Figure 7). 1H-NMR and 13C-
on the results of the spectrum analysis NMR chemical shift data obtained from
obtained, it is known that the secondary this study with comparisons can be seen
13
metabolite compounds resulting from in Table 1. Analysis of the C-NMR
isolation are steroid class compounds, in spectrum (125 MHz, CDCl3) shows that
the presence of the OH group and the the isolated steroid compound has 28
unconjugated C=C double bond group carbons. From the DEPT-135 analysis
which is the characteristics of steroid with the help of the HSQC analysis, it is
compounds. known that there are six methyl
Nuclear magnetic resonance carbons, eight methylene carbons,
spectroscopy or NMR spectroscopy was eleven methine carbons, and three
used in this study, namely, 1H-NMR and quaternary carbons
13
C-NMR. Chemical shift data of 1H-
Table 1. 1H-NMR data (500 MHz, CDCl3) and 13C-NMR (125 MHz, CDCl3) of
isolated compounds and comparative literature data from Brassicasterol
(Jinming et al., 2001)

Secondary Metabolite Brassicasterol

No
Δc (ppm) DEPT HSQC HMBC Δc (ppm) δH (ppm)
1 37,3844 CH2 1,8524 (H1) 71,9498 (C3) 37,54
1,2485 (H2)
2 29,0823 CH2 31,95
1,7015 (H2)
3 71,9498 CH-OH 3.5219 (H3) 72,04 3,53
4 42,3455 CH2 2,274 (H4) 71,9498 (C3) 42,58
140,8764 (C5)
121,8667 (C6)
 31, 7851 (C7)
36,6485 (C10)
5 140,8764 C - 141 -
42,3455 (C4)
6 121,8667 CH 5.3496 (H6) 31,7851 (C7) 121,9
36,6485 (C10)
1,8160 (H7)
7 31,7851 CH2 32,16
1,9752 (H7)
8 32,0291 CH 1,4492 (H8) 21,23 (C11) 33,34
9 51,3804 CH 1,5253 (H9) 32,029 (C8) 50,51
10 36,6485 C - 36,78
11 21,3633 CH2 1,4909(H11) 56,9978 (C14) 21,32
12 39,8087 CH2 1,9752 (H12) 39,96
13 42,4267 C - 42,58 -
14 56,9978 CH 1,1556 (H14) 12,1883 (C18) 57,12
15 24,5071 CH2 1,5447 (H15) 32,0291 (C8) 24,51
32,0291 (C8)
1,4503 (H16)
16 25,5654 CH2 42,4267 (C13) 28,68
1,1713 (H16)
12,1883 (C18)
12,1883 (C18)
17 56,0669 CH 1,1516 (H17) 56,36
21,3633 (C11)
39,8087 (C12)
18 12,1883 CH3 0.6940 (H18) 12,3 0,69
42,4267 (C13)
19 21,2069 CH3 0,8362 (H19) 51,3804 (C9) 20,12 0,83
20 40,6686 CH 2,0264 (H20) 40,26
40,6686 (C20)
21 21,2531 CH3 1,0244 (H21) 138,4801 (C23) 21,28 1
24,5071 (C15)
40,6686 (C20)
22 129,3881 CH 5.0171 (H22) 138,4801 (C23) 136,1 5,2
50,2728 (C24
40,6686 (C20)
23 138,4801 CH 5.1396 (H23) 129,3881 (C22) 132 5,17
50,2728 (C24)
24 50,2728 CH 0,9362 (H24) 43,06
25 32,0291 CH3 1,5253 (H25) 50,13 (C24) 33,34
26 19,5492 CH 1,0075 (H26) 50,2728 (C24) 19,84
27 19,1205 CH3 0,7855 (H27) 19,58
28 12,4127 CH3 0.7944 (H28) 32,0291 (C25) 17,8
Figure 4. The HSQC correlation spectrum for the isolated compound
Analysis based on literature it is known that there is one hydroxy
obtained 1CNMR, DEPT, and HSQC group from pure isolate that appears in
data, it is known that there is one signal the chemical shift of 71.9498 ppm. It is
C atom which is estimated to overlap also known that two double bonds give
with each other. This overlap signal is rise to a carbon signal in the chemical
estimated between C-8 and C-25. This shift of 121.8667 ppm, 129.3881 ppm,
is because the chemical environment of 138.4801 ppm, and 140.8764 ppm
the two is almost the same. In addition (Figure 4.).
to the known overlap in C-8 and C-25,
Figure 5.Correlation spectrum of HMBC for Isolated Compound

The HMBC data above strengthens Figure 6. The chemical shift analysis of
the positioning of each carbon that has the data for the isolated steroid
been known to be correlated from the compounds is quite similar to the
HSQC data that has been analyzed comparative data. The structure of the
previously. In Figure 5, it can be seen brassicasterol compound can be seen in
that C3, which is the carbon that binds Figure 7. The brassicasterol compound
the hydroxyl group, is correlated with has a similar structure to the isolated
H1. Figure 5 depicts H6 which is a stigmasterol compound (Rohmawati &
proton for one of the vinyl groups Sutoyo, 2018) from Pometia pinnata.
correlating with C4, C7, and C10. C23 Structurally, these two compounds are
carbon which is the carbon for the vinyl similar, namely the location of the vinyl
group has a correlation with H23 and groups at positions C5, C6, C22, and
the proton H22 which is one of the C23. The location of the -OH group of
protons that binds to the carbon making these two compounds also has
up the vinyl group correlates with C20, similarities where both are at C3. The
C23, and C24. Figure 5 shows a thing that distinguishes these two
correlation between H4 and C5 and C6. compounds is the amount of carbon.
There is also a correlation between H22 Brassicasterol has a total carbon of 28
and C23, H23 and C22 while the stigmasterol compound has a
The data above can also be carbon of 29 where the carbon is
simulated the correlation of HMBC in located at C26.

21 25
22
20
18 24
23
12 27
14 26
11 13
19 15
28
1 9 17
16
2 10 8

3 5 7
HO 4 6

Figure 6. HMBC Correlation Figure 7. Structure of Brassicasterol ((22E,

24R) -Ergosta-5, 22-dien-3b-ol)
CONCLUSION shrimp toxicity of various
The compound isolated from the leaves polarities leaves and fruits crude
of Pometia pinnata is thought to be a fractions of Ziziphus jujuba native
steroid class compound, namely to Oman and their antimicrobial
brassicasterol. This is supported by the potency. Sustainable Chemistry
carbon and proton analysis of NMR, and Pharmacy, 5(January), 122–
DEPT, HSQC, and HMBC showing a 126.
spectrum consistent with the literature. Alegantina, S., & Isnawati, A. (2010).
The cytotoxic test results on the n- Identifikasi dan penetapan kadar
hexane, dichloromethane, ethyl acetate, senyawa kumarin dalam ekstrak
and methanol fractions showed that the metanol. Buletin Penelitian
n-hexane fraction was more active with Kesehatan, Vol. 38, pp. 17–28.
a value of LC50419.855 mg / L. Gelani, C. D., & Uy, M. M. (2016).
However, the cytotoxic properties of the Cytotoxicity to Artemia salina L.
brassicasterol compound that have been of marine sponge extracts from
isolated have not been carried out due to Surigao del Norte, Philippines.
the small number of samples. It is hoped Bulletin of Environment,
that later tests will be carried out for the Pharmacology and Life Sciences,
cytotoxic properties of this isolated 5(April), 14–18.
brassicasterol compound Jinming, G., Lin, H., & Jikai, L. (2001).
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