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GreenVC IOP Publishing
IOP Conf. Series: Earth and Environmental Science 160 (2018)
1234567890 ‘’“” 012005 doi:10.1088/1755-1315/160/1/012005

Isolation, identification of alkaloid from Rhizophora

mucronata and the activity of its methanol extract against
barnacles

D G S Andayani1*, S R Anggraeni2, E Liviawaty2, R M Chrisentia2 and Y

Srikandace1
1
Research Unit for Clean Technology, Indonesian Institute of Sciences (LIPI),
Komplek LIPI Bandung, Jalan Sangkuriang, Gedung 50, Bandung, 40135, Indonesia
2
Faculty of Fishery and Marine Science, Padjadjaran University, Jl. Raya Bandung
Sumedang KM 21, Jatinangor 40600, Indonesia
*
Email: [email protected]

Abstract. A study was conducted to isolate, identify alkaloid content of Rhizophora mucronata
and to determine its effectiveness of methanol extract against barnacles. The use of tributyltin
(TBT) was effective to reduce biofouling. However, TBT has been prohibited because they are
detrimental to non-target organisms and for surrounding environment. Rhizophora mucronata is
useful as antifouling compounds because of the secondary metabolites. Its active compound will
replace TBT as it is safe against the biota and the marine environment. The root's bark, trunk's
bark and leaves powder were macerated using methanol. The methanol extract was used for total
alkaloid isolation. The separation of alkaloids was done by preparative TLC and analyzed with
FTIR and LC-MS. The results showed that the alkaloid metabolite identified was benzamide
(C17H14N2O2). The methanol extract had antifouling activity against barnacles. The highest yield
was found in Rhizophora mucrophora leaf extracts at about 24.8%. Antifouling activity test
against barnacles showed that a mixture of wood paint and Rhizophora mucronata leaf, root and
bark extracts were not significantly different.

1. Introduction
Submarine objects, especially in coastal waters, are attached by biofouling organisms. Barnacle is a
macrofouling that forms a fouling community causing damage to beach buildings and increase fuel
consumption, damage to pipes, pumps and vessels can be reversed in bad weather conditions [1,2].
Biofouling or biological fouling is the accumulation of microorganisms, plants, algae or animals on
wetted surfaces [3].
Antifouling agent is a substance that prevents or retards fouling or marine underwater growth on
plants, rocks or ships’ bottoms [3]. Currently fouling prevention is done using antifoulant paint, which
active compound is tributyltin (TBT). This compound is harmful to organisms. The copper content in
TBT and continuous accumulation of TBT will poison the organisms in the sea, causing impaired
immune systems and shells to form changes such as malformation [4,5].
The greatest interest in the use of natural products as antifoulants is based on their potential to
function as nontoxic antifoulants. The new diterpene methoxy-ent-8 (14)-primarenely-15-one (1) and
three metabolites: ent-8(14)-primarene-15R, 16-diol (2), stigmasterol (3) and β-sitosterol (4) were
isolated from the roots of mangrove plant Ceriops tagal [6-8]. The objective of this research was to

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GreenVC IOP Publishing
IOP Conf. Series: Earth and Environmental Science 160 (2018)
1234567890 ‘’“” 012005 doi:10.1088/1755-1315/160/1/012005

isolate and identify alkaloids in Rhizophora mucronata extract derived from leaves, stems and roots, as
well as to analyze the activity of the methanol extract as antifouling agent.

2. Methods
2.1. Extraction and fractionation
Isolation and fractionation of alkaloid metabolites was done from the roots, stems and leaves powder of
mangrove. Extraction was carried out by maceration with methanol then concentrated using a rotary
evaporator. The methanol extract was added with HCl 1 N, then extracted with ethyl acetate. The acid
layer was added with ammonium hydroxide then re-extracted with ethyl acetate. The ethyl acetate layer
was concentrated with a rotary evaporator to obtain a total alkaloid extract [9,10].

2.2. Separation, purification, and identification

The total alkaloid was purified by thin layer chromatography (TLC) and eluted with methanol, ethyl
acetate, and hexane using silica gel plate 60 GF254. The spots were observed under UV lamp at 366 nm.
Further separation was done using preparative TLC to obtain the pure alkaloid. The alkaloid isolate was
analyzed and identified by FTIR and LC-MS [11,12].

2.3. Testing of alkaloid metabolite

The alkaloid of roots, barks and leaves of Rhizophora mucronata was determined with Dragendorff’s
test. Two mg of methanol extract was acidified with 2 M HCl and added by 1 mL of Dragendorff’s
reagent. Formation of orange or orange red precipitate indicated the presence of alkaloids. Wagner’s test
was done by preparing 2 mg of methanol extract, acidified with 1.5 % v/v of HCl, and added with several
drops of Wagner’s reagent. A yellow or brown color indicated the presence of alkaloids. Furthermore,
the extract was added with HCl and NaOH followed by extraction with ethyl acetate.

2.4. Antifouling activity determination

The antifouling activity test was conducted in the sea waters of Teluk Awur Beach, Jepara, Central Java
Indonesia. Temperature, pH, salinity, and brightness were recorded during the experiments for 16 days.
Antifouling activity was observed by counting the number of macrofouling barnacle which attached on
wooden board media. A 7 x 14 cm2 area of wooden board was painted using a commercial paint mixed
with extract of roots, stems or leaves of mangrove at 500 or 1000 ppm concentration. As a positive
control used was wooden board of the same size painted with commercial antifouling, whereas the
negative control used was the same commercial-painted wooden board without mixing with neither
mangrove extract nor plain wood planks. The wooden boards were weighted and tied to a pivot on a
dock and were spaced 50 cm from the sea level at the lowest tide; the distance between the boards and
the beach was about 12 m [13-15].

2.5. Data analysis

The data was analyzed descriptively by standard deviation (STDEV) based on the number of attachment
of macrofouling biota on wood board media. The standard deviation is a measure of how widely values
are dispersed from the average value (the mean). The experiment was repeated three times. The standard
deviation was calculated using the ”n-1” method with the following formula:
√Σ(x − x̅ )2
(n − 1)

3. Results and discussion

3.1. Extraction and fractionation

Extraction by methanol submergence method was intended to bind the polar compounds. Alkaloids are
polar compounds that have to be extracted with polar reagent. Methanol is the most polar reagent after
water. The rendement of mangrove extract is shown in Table 1.

2
GreenVC IOP Publishing
IOP Conf. Series: Earth and Environmental Science 160 (2018)
1234567890 ‘’“” 012005 doi:10.1088/1755-1315/160/1/012005

Table 1. Rendement of root’s bark, stem’s bark and leaves extracts of Rhizophora
mucronata.
Sample weight (gr) Extract weight (gr) Rendement
Sample
(%)
Root’s bark 401.6 69.7 17.4
Stem’s bark 500.1 89.2 17.9
Leaves 387.2 96.0 24.8

Rendement is a comparison between the weights of the ingredients used with the total weight of
the material. The value of rendement is used to determine the effectiveness of bioactive metabolite
material. The higher the yield value indicated the higher the value of the methanol extract produced. The
highest rendement value was obtained from the leaves extract of Rhizophora mucronata because the
leaves had smoother texture compared with stem’s and root’s barks. The tiny size of the leaves extract
is easy to withdraw the compound. It correlates with the highest activity against macrobiota
[7,8,11,16,17].

3.2. Alkaloid test of mangrove extract

The methanol extracts of roots, stems and leaves of Rhizophora mucronata showed positive alkaloid
reactions proven by white deposits when Mayer and Dragendorff reagents were added (Table 2).

Table 2. Testing of Alkaloid metabolite of Rhizophora mucronata.

Root’s bark Stem’s bark Leaves
Alkaloid
Methanol Methanol Methanol
metabolite Simplicia Simplicia Simplicia
extract extract extract
Sample + (Mayer
+ Dragendorff) + + + + + +
reagent
+ = contains metabolite alkaloids

3.3. Separation and purification

The separation showed three spots of TLC. The ratio of eluent was 21 methanol: 6.5 ethyl acetate: 5.5
n-hexane. The results showed that the topmost spot of a reddish-purple shine indicated an alkaloid
compound. The Rf spot was 0.9 (Figure 1).

Figure 1. TLC of alkaloid isolate.

Separation of alkaloid compounds with preparative TLC was performed two yellow and one dark
green bands. On the yellow bands are scraped subjected for purity by TLC again using the same
developer.

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GreenVC IOP Publishing
IOP Conf. Series: Earth and Environmental Science 160 (2018)
1234567890 ‘’“” 012005 doi:10.1088/1755-1315/160/1/012005

Figure 2. FTIR spectogram of alkaloids of mangrove leaves extract.

Figure 3. Spectogram of LC-MS.

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GreenVC IOP Publishing
IOP Conf. Series: Earth and Environmental Science 160 (2018)
1234567890 ‘’“” 012005 doi:10.1088/1755-1315/160/1/012005

3.4. Identification of compounds

Based on FTIR spectroscopy analysis, the alleged alkaloid compounds contained in the mangrove leaves
were N-H, NH, C-O, C-N, CH and C-C groups. FTIR spectogram of alkaloids isolated from the leaves
extract of Rhizophora mucronata is presented in Figure 2.
The results of FTIR spectroscopy showed the functional groups of the isolate samples. Figure 2
shows the vibration peaks with an absorption at the wavelength of 3444.87 cm-1, which was the
absorption of the vibration stretching of the NH group. It was also supported by the absorption at the
wavelength of 1570.06 cm-1, which was the vibration buckling of the NH group. The wavelength of
1051.20 cm-1 was a buckling vibration of C-O. The presence of vibration in the wave number 1382.96
cm-1 was the absorption of C-N, while at 2432.24 cm-1 wavelength was the bending vibration of CH and
at 1633.71 cm-1 was the vibration of C-C group.
The isolate was analyzed using Liquid Chromatography-Mass Spectroscopy (LC-MS) to determine
the molecular weight of the alkaloids. Spectogram of the LC-MS is shown in Figure 3.
The molecular weight of the isolated compound indicated by the LC-MS spectrometer was 278
g/mol. It is shown in Figure 3 in the presence of m/z 279.2093 [M + H]+ and m/z 301.1956 [M+Na]+.
Based on these results, the isolate of alkaloids from mangrove leaf contained a benzamide compound,
N-1-isoquinolinyl-3methoxy- with the chemical formula C17H14N2O2. The chemical structure of the
benzamide alkaloid (C17H14N2O2) is shown in Figure 4.

Figure 4. Alkaloid benzamide.

Six new 4-phenyl-3,4-dihydroquinolone derivatives along with the related of inquinolone 4 were
isolated and identified from the cultures of an endophytic fungus obtained from the fresh leaves of the
marine mangrove plant Rhizophora stylose [18]. Many bioactive metabolites including
anthracenediones, xyloketals, sesquiter penads, chromones, lactones, coumarin and isocoumarin
derivatives, xanthones and peroxides have been isolated from various mangrove-derived fungi in the
South China Sea [19]. The methanol extract of Rhizophora mucronata contained alkaloid, flavonoid,
and tannin [16], seven compounds were identified in the fraction of ethyl acetate of Rhizophora
mucronata stem bark namely quinone, steroid, alkaloid and aromatic group with the highest secondary
metabolite content was alkaloid group (74.8%) [20]. 2-isocyanato-2-hexanamine that was successfully
isolated from mangrove plant was active against bacterial disease in shrimp [21]. The compounds
contained in the skin of the mangrove stem Sonneratia alba is a phenolic group with lactone rings [22],
the main secondary metabolites in mangrove plant in the form of alkaloid class of polar compounds. The
white crystalline powder form characteristics had a melting point of 172°C with molecular weight of
232, and the molecular formula was C12H12N2O3 [23].

3.5. Antifouling activity

The data obtained was in the form of the number of attachment of macrofouling biota on wood
board media. The data was analyzed descriptively with three replicates based on related sources in the
form of journals, books, and thesis and presented in tabular form. The antifouling activity of Rhizophora
mucronata extract at 500 and 1000 ppm was not significantly different with the amount of attachment
between 247-315 barnacles (Table 3).

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GreenVC IOP Publishing
IOP Conf. Series: Earth and Environmental Science 160 (2018)
1234567890 ‘’“” 012005 doi:10.1088/1755-1315/160/1/012005

Table 3. Barnacles attachment.

Sample Concentration of extract (500 ppm) Concentration of extract (1000 ppm)
Wood+paint+
extract
(Rhizophora
Root skin Bark stem Leaf Root skin Bark stem Leaf
mucronata)
Barnacles 280±35.68 315±15.95 253±42.74 282±23.67 286±47.51 247±66.16
attachment a a a a a a
(individual)

Table 4. Barnacles attachment (control).

Control Standard Barnacles attachment
(individual)
Positive (+) Wood + commercial antifouling paint 4 ± 3.0a
Wood + paint without antifouling 395 ± 60.68c
Negative (-)
Wood without painting 190 ± 98.38b

The results indicated that concentration of the extracts did not affect barnacles attachment. All
values were not statistically different (Table 3).

3.6. The effectiveness of antifouling extract of Rhizophora mucronata

The bacteria initiate the attachment and form a primary thin film on the surface of submerged objects.
The formed thin layer allows benthic diatoms, algae spores and larvae of various types of aquatic animals
to attach and grow on the surface of the object. At the end, cauced bryozoa, hydrozoa, ciripedia, tunikata,
algae and other biota to be a group of biota that developed later after forming layer film by bacteria.
Rhizophora mucronata extract inhibited the growth of biofilm bacteria that reduced macrofouling
attachment [17].
The number of barnacles attached to the wood media was due to the wood media surface was
uneven and had many gaps so that the barnacles were attached to the cracks on the wooden surface.
Selection of wood species for the test board was based on the type of wood commonly used in the
manufacture of boats e.g. teak, meranti, and camphor. Meranti wood was selected because the price was
more economical and easily obtained [24].
In addition to media and habitats, the attachment rate of barnacles was influenced by brightness,
temperature, and current velocity [25]. Barnacles grow well at depth and brightness between 20-130 cm
[26]. Water temperature during observation was 27°C on average; the temperature was quite good for
the growth of barnacles because barnacles like waters with temperature of 15-35°C [26]. Soluble salinity
or salinity in the study area was 36 ppt, the salinity was particularly suitable for growing barnacles
because barnacles are able to live in estuary waters and have a salinity tolerance between 15-41 ppt [26].
The degree of acidity or pH of the waters at the time of observation was very good that reached 8,
barnacles can live optimally at a pH between 6-9 [27].
Based on the number of wooden barnacles attached to wood with wood paint mixture, Rhizophora
mucronata 1000 ppm leaf extract was a more effective anti fouling than root bark extract. However, the
mixture of wood paint and Rhizophora mucronata 1000 ppm leaf extract would be much less effective
when compared with commercial antifouling paint because the concentration of the active ingredients
or secondary metabolites in commercial paints was very high compared with the active ingredients in
mangrove leaf extract, due to the copper content.

4. Conclusion
Mangrove leaf extract (Rhizophora mucronata) contained betanidine alkaloid compounds (C17H14N2O2)
and showed an antifouling activity against barnacles.

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GreenVC IOP Publishing
IOP Conf. Series: Earth and Environmental Science 160 (2018)
1234567890 ‘’“” 012005 doi:10.1088/1755-1315/160/1/012005

Acknowledgement
The authors would like to thank the management of Teluk Awur Beach, Jepara, Central Java Indonesia
for allowing us to conduct antifouling activity test. The research was funded by Mandiri Research of
Indonesian Institute of Sciences (LIPI).

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