Chiang Mai J. Sci. 2014; 41(1)
97
Chiang Mai J. Sci. 2014; 41(1) : 97-104
http://epg.science.cmu.ac.th/ejournal/
Contributed Paper
Potential Pharmaceutical Uses of the Isolated
Compounds from Silkworm Excreta
Sornkanok Vimolmangkang, Chanida Somkhanngoen and Suchada Sukrong*
Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences,
Chulalongkorn University, Bangkok 10330, Thailand.
*Author for correspondence; e-mail: [email protected]
Received: 18 November 2012
Accepted: 27 May 2013
ABSTRACT
Silkworm (Bombyx mori) excreta have long been used in the pharmaceutical and food
industries as a natural colorant. In some countries, instant silkworm tea is available due
to its traditional Eastern medicine health benefits. However, there are limited data on
the identification of bioactive compounds in silkworm excreta. Here we have isolated
and identified for the first time in silkworm excreta 1-tritriacontanol, a very long chain
fatty alcohol, and lupeol, a phytosterol, in addition to β-sitosterol. The compounds
were isolated from a crude acetone extract, purified by vacuum liquid column
chromatography, and partitioned by hexane and ethyl acetate. The structure elucidation
was confirmed by means of fourier transformed infrared spectroscopy (FT-IR), proton
and carbon nuclear magnetic resonance (NMR), and electron ionization mass spectrometry
(EIMS). The cytotoxic activity of the crude extract and 1-tritriacontanol were assayed
against vero cells and different human cancer cell lines and they were found to be inactive,
demonstrating their safety. These data support the use of silkworm excreta in traditional
medicine and show the potential of silkworm excreta as a novel natural source of very
long chain fatty alcohols.
Keywords: silkworm excreta, silkworm feces, Bombyx mori, lupeol, tritriacontanol,
very long chain fatty alcohol, cytotoxicity
1. INTRODUCTION
Silkworm excreta or feces are excreted
from silkworm (Bombyx mori) and considered
to be a major waste product of sericulture
(silkworm cultivation). However, silkworm
excreta have both pharmaceutical and food
industrial uses. In traditional medicine,
silkworm feces have been used as a therapeutic
agent in China, Korea and some Eastern Asian
countries to treat infectious diseases, headache
and abdominal pain [1]. Moreover, silkworm
excreta are a good source of natural colorant
for the food industry. Silkworm excreta are
also available in the form of an instant tea,
which is claimed to be a nutraceutical product.
Although silkworm feces have had
medicinal uses for a long time, the published
reports on the isolation of the chemical
compounds and their structure elucidation are
limited. Silkworm fed on mulberry leaves
were found to excrete more than half of the
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leaves without digestion [2], suggesting that
some of the compounds in the excreta
are from mulberry leaves and some are
biotransformed in the silkworm intestine.
Physically, silkworm excreta have a cylindrical
shape of 2-3 mm in length with a deep
green color. The chemical constituents of
silkworm excreta that have principally been
reported are chlorophyll and chlorophyll
derivatives, xanthophyll, carotenoid,
and flavonoids [3, 4]. The lipid profile of
silkworm excreta has also been investigated
[5]. Group compositions of the lipids were
reported in silkworm excreta in which there
were concentrated liposoluble compounds
such as phytosterol, unsaturated fatty acid,
and fatty alcohol. However, identification
of liposoluble compounds in the excreta is
very limited. Only triacontanol, a very
long chain fatty alcohol (VLCFA), and
β-sitosterol, a phytosterol, were reported in
silkworm excreta [6].
This study was focused on the isolation
of bioactive compounds from silkworm
excreta that are particularly solubilized in
hydrophobic solvent in order to investigate
the presence of liposoluble bioactive
substances. Identification of compounds
found in silkworm feces may validate its
traditional use and lead to a new use in
pharmaceutical and food industries. Here we
have identified a VLCFA, 1-tritriacontanol,
and two phytosterols, lupeol and β-sitosterol.
To our knowledge, this is the first time that
1-tritriacontanol and lupeol have been
isolated from silkworm excreta. Chemical
structures of these three compounds are
shown in Figure 1.
Chiang Mai J. Sci. 2014; 41(1)
Figure 1. Structure of three isolated
compounds: a 1-tritriacontanol (SW1); b
lupeol (SW2); and c β-sitosterol (SW3).
Carbon positions were labeled.
2. MATERIALS AND METHODS
2.1 Sample Preparation
Feces from various silkworm (Bombyx
mori) stages were obtained from a local
sericulture community (Ubonratchathani
Province, Thailand). Samples were air-dried
by local farmers to protect against microbial
deterioration before being given to the
researchers. Once received, visible foreign
matters such as small pieces of plant leaves
and dead silkworm bodies were manually
removed. Samples were dried at 60°C for
5 h and then ground into a fine powder.
Otherwise, fresh samples were kept at -20°C
if extraction was not performed on the day
of delivery.
2.2 Extraction and Isolation
The crude acetone extract of silkworm
excreta (1.6 g) was separated by vacuum liquid
column chromatography using a sintered
glass filter column of silica gel (No. 7734,
112 g). Eighty-one fractions were collected
after elution from the column using mixtures
of hexane and ethyl acetate (from 0% ethyl
acetate in hexane to 100% ethyl acetate) in a
Chiang Mai J. Sci. 2014; 41(1)
polarity gradient manner. The fractions were
combined to yield 14 fractions (fraction A
to N) by following similar thin layer
chromatographic patterns. Three compounds,
SW1 - SW3, were isolated.
The purification steps are illustrated in
Figure 2. Briefly, fractionation of fraction F
(189.3 mg) was performed on a silica gel
(No. 9385) column by elution with mixtures
of hexane and dichloromethane to give 12
fractions (F1-12). To isolate the compounds,
fraction F7 and F8 were eluted separately in a
polarity gradient manner with mixtures of
hexane and ethyl acetate (from 5% to 15%
ethyl acetate in hexane) to give compound
SW1 and compound SW2, respectively.
Compound SW3 was isolated from fraction
G by first partitioning with methanol and ethyl
acetate (0% to 50%) and then with hexane
and ethyl acetate (7:3). The compounds were
then further identified their structure.
Figure 2. Scheme of purification steps for
compound a 1-tritriacontanol (SW1), b lupeol
(SW2), and c b-sitosterol (SW3). The main
fractions were highlighted in bold fonts and
lines. Abbreviations are followed. VLC:
vacuum liquid chromatography; FCC: flash
column chromatography; CC: column
chromatography; EtOAc: ethyl acetate;
CH2Cl2: chloroform; MeOH: methanol.
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2.3 Structure Elucidation
The isolated compounds were identified
by fourier transformed infrared spectroscopy
(FT-IR), proton and carbon-13 nuclear
magnetic resonance (1H and 13C NMR,
respectively) spectroscopy, and electron
ionization mass spectrometry (EIMS). FT-IR
spectrum was recorded on a Spectral Perkin
Elmer Spectral one (PerkinElmer, MA, USA).
1
H NMR (300 MHz) and 13C NMR (75 MHz)
spectra were obtained using a Bruker Avance
DPX-300 FT-NMR spectrometer (Bruker
Biospin, Fallanden, Switzerland). Deuterated
chloroform (CDCl3) was used as the solvent
for NMR. Chemical shifts were recorded in
ppm scale using the chemical shift of the
solvent as the reference signal. Coupling
constants (J) were reported in Hertz (Hz).
Electron ionization mass spectra were
acquired using a Micromass LCT
spectrometer or a Thermo-Finnigan Polaris
Q mass spectrometer (Waters Corporation,
MA, USA).
2.4 Cytotoxicity Assay
The cytotoxic activity of the crude extract
and 1-tritriacontanol was conducted at the
National Center for Genetic Engineering
and Biotechnology, Thailand. The crude
extract (25 mg/ml) and the 1-tritriacontanol
(50 mg/ml) were tested for their cytotoxicity
against different cancer cell lines [KB (Oral
cavity cancer), MCF7 (breast cancer), and
NCI-H187 (small cell lung cancer)] using a
resazurin microplate assay (REMA) and
against vero cells (African green monkey
kidney) using a Green Fluorescent Protein
(GFP)-based assay. Ellipticine and 0.5%
DMSO were used as positive and negative
controls, respectively. For the cancer cell lines,
activity was interpreted from %inhibition
where less than 50% was considered inactive.
For the vero cells, %cell growth was used;
a cell growth rate of ≥50% was interpreted
100
to mean that the compound was noncytotoxic.
3. RESULTS AND DISCUSSION
3.1 Identification of Bioactive
Compounds in Silkworm Excreta
Isolation of compounds from a crude
acetone extract of silkworm excreta was
performed by vacuum liquid column
chromatography following separation by
several solvent mixtures in a polarity
gradient manner (Figure 2.). Eventually, three
compounds designated SW1, SW2, and SW3
were purified. From 1.6 g of crude extract,
7.5 mg SW1, 15.9 mg SW2, and 3.1 mg SW3
were obtained, which were approximately
4.68, 9.94, and 1.94 mg per gram crude
extract, respectively. The structural information
of the compounds was elucidated by FT-IR,
1
H and 13C NMR spectral data and mass
spectra (MS) fractionation patterns.
Compound SW1 was identified as
1-tritriacontanol, which had not previously
been isolated from silkworm excreta and is
not present in mulberry leaves. Compound
SW2 was identified as lupeol, which is
normally found in mulberry leaves [7] but
has not previously been isolated from
silkworm excreta. Compound SW3 was
identified as β-sitosterol, which was previously
reported in both silkworm excreta and
mulberry leaves [6, 7].
Compound SW1 appeared as a white
solid compound. 1H NMR (CDCl3) δ: 3.66
(2H, t, J = 6.6 Hz), 1.58 (2H, bs, J = 6.3 Hz),
1.27 (60H, m), 0.87 (3H, t, J = 6.9 Hz).
The 13C NMR (CDCl3) δ: 63.11, 32.8, 25.7,
29.6 (C4-C30), 32, 22.6, 14. IR (KBr) cm-1:
3412, 2849, 2920. MS m/z: 480 (M+) (Calcd
for C33H68O). Overall, compound SW1 was
identified as 1-tritriacontanol by comparing
the 1H and 13C NMR, IR, and MS data to
those data reported in the literature [8].
Compound SW2 appeared as white
Chiang Mai J. Sci. 2014; 41(1)
needles that were positive to anisaldehyde
detection. Structure elucidation identified
compound SW2 as lupeol. The 1H and 13C
NMR spectra of compound SW2 were
compared to those of lupeol previously
reported in [9]. 1H NMR (CDCl3) δ: 4.70 and
4.59 (each 1H, s), 3.22 and 3.19 (1H, dd, J=
5.4, 6.6 Hz), 2.38 (1H, m), 1.70 (3H, s), [1.05,
0.99, 0.96, 0.85, 0.81, and 0.78] (each 3H, s).
13
C NMR (CDCl3) δ: 150.7, 109.3, 78.8, 55.3,
50.5, 48.3, 48.0, 43.0, 42.8, 40.8, 40.0, 38.7
(C1, C4), 38.1, 37.1, 35.6, 34.3, 29.9, 28.0, 27.5,
27.4, 25.2, 21.0, 19.3, 18.3, 18.0, 16.1, 16.0,
15.4, 14.6. MS m/z: 426 (M+) (Calcd for
C30H50O).
Compound SW3, which appeared as a
white powder, gave a purple spot when
anisaldehyde TS was applied, indicating a
steroidal or triterpenoid skeleton. The
structure of compound SW3 was elucidated
and identified as β-sitosterol through
comparison of its 1H and 13C NMR data with
previous reported values [10]. 1H NMR
(CDCl3) δ: 5.37 (1H, d, J = 5.1 Hz), 3.54 (1H,
m), 2.31-1.20 (30H, m), 1.05 (3H, s), 0.970.79 (3Hx4, m), 0.69 (3H, t, J = 6 Hz). 13C
NMR (CDCl3) δ: 140.77, 121.71, 71.82, 56.79,
56.0, 45.88, 42.33, 39.8, 37.27, 36.52, 36.15,
33.98, 31.92, 31.69, 28.24, 26.15, 24.3, 23.1,
21.1, 19.8, 19.39, 19.04, 18.78, 11.99, 11.86
(C18, C25). IR (KBr) cm-1: 3434, 2959, 2869,
1634, 1465, 1383. MS m/z: 414 (M+) (Calcd
for C29H50O).
3.2 Silkworm Excreta: A Potential Source
of Very Long Chain Fatty Alcohols
Triacontanol, which is a long chain fatty
alcohol containing 30 carbon atoms, has
previously been isolated from silkworm
excreta [6]. However, no other VLCFAs have
ever been isolated despite the fact that the
lipid profile of silkworm excreta suggested
that fatty alcohols and fatty acids were found
in the second to the highest proportion of
Chiang Mai J. Sci. 2014; 41(1)
the other liposoluble components [5]. In this
study, we isolated 1-tritriacontanol, which is a
VLCFA containing 33 carbon atoms. To our
knowledge, this is the first time that it has been
isolated from silkworm excreta. Although
VLCFAs are usually present in plant wax and
beeswax, odd-numbered carbon fatty alcohols
such as tritriacontanol do not seem to be
common VLCFAs in plant. A few reports
had identified tritriacontanol in some medicinal
plants such as Achyranthes aspera [8] and
Chloroxylon swietenia [9] but it has never been
reported in food crops such as wheat,
sugarcane, maize and in other fruits, seeds, and
nuts. Waxes from those food crops primarily
contain C26-30 alcohols [11, 12]. In addition,
tritriacontanol has not been reported so far in
mulberry leaves, the only food source for
silkworm in the study. In the synthesis of oddchain fatty acids, propionate (C3) is utilized as
a substrate [13] and it has been found that
propionate was abundantly produced through
bacterial fermentation of ingested plant in the
rumens of animals. Thus, it is more likely that
tritriacontanol is produced by microbes within
the silkworm intestine.
Silkworm excreta have been used to
lower blood cholesterol in traditional
medicine. Clinical studies have shown that it
lowers cholesterol levels and blood pressure
[14]. This biological activity would be due to
VLCFAs present in silkworm excreta.
According to numerous studies, consumption
of VLCFAs decrease LDL cholesterol and
increase HDL cholesterol in humans [14].
Thus, identification of tritriacontanol in this
study provides evidence of the presence of
VLCFA in addition to the previously reported
triacontanol.
Recently, public interest in the use of
VLCFAs as a human dietary supplement to
help lower blood cholesterol has been
increasing. Policosanol, which is commonly
found in dietary supplement form, is a mixture
101
of VLCFAs (20-36 carbons) isolated from
plant waxes such as sugarcane wax, spinach
wax, or beeswax [12, 14]. Several studies
have reported that policosanol was able to
lower cholesterol in various patient groups
with elevated cholesterol such as the middleaged patients with non-insulin-dependent
diabetes mellitus [15]. The principal source of
commercially used policosanol is sugarcane.
Here, we found that silkworm excreta may
be a new source of VLCFAs for the following
reasons: first, similar to sugarcane, they
contain a high proportion of various fatty
alcohols [5]; secondly, at least two VLCFAs,
triacontanol and tritriacontanol, were
identified; and thirdly, silkworm excreta are
abundant and collection is extremely quick
and easy. In addition, unlike sugarcane, they
do not require time to grow and they are a
continuously produced by-product of the
sericulture industry. However, the isolation
and identification of other long chain fatty
alcohols, particularly docosanol (C22),
tetracosanol (C24), hexacosanol (C26), and
octacosanol (C30), in silkworm excreta should
be further studied in order to strengthen the
possibility of using silkworm excreta as a new
source of VLCFAs for commercial use.
3.3 Anti-Inflammatory Activity of
Silkworm Excreta May Result from
Lupeol and β-Sitosterol
Lupeol and β-sitosterol are terpenoid
compounds that are found in various plant
species. In the present study, they have
been found in silkworm excreta. Although
β-sitosterol was previously identified in
silkworm excreta [6], lupeol has been found
for the first time in this study. However,
lupeol and β-sitosterol would not be
produced from silkworm itself but from
mulberry leaves (Morus alba L.), which is the
only food source for silkworm. Previously,
both of them have been identified in
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mulberry extract [7]. It has been reported
that more than half of mulberry leaf-intake
by silkworm is excreted without being
digested [2]. Accordingly, the presence of
lupeol and β-sitosterol in silkworm excreta
suggests that these compounds are more
likely to be derived from mulberry leaves that
are not processed in the silkworm intestine
and are thus excreted in the feces.
Traditionally, silkworm excreta have
been used in treatment of abdominal pain
suggesting its analgesic and anti-inflammatory
effects. In addition, crude extract of silkworm
feces used in traditional Chinese medicine
has shown anti-inflammatory activity in
carrageenan-induced edema rats [16]. Recently,
flavonoids newly isolated from silkworm
droppings exhibited an anti-inflammatory
activity via a suppression of inflammatory
mediators [4]. However, lupeol isolated from
feces in the present study may be partly
responsible for this activity. The antiinflammatory effect of lupeol has been
reported in a number of studies. A strong
anti-inflammatory effect was observed in
rat liver cells where lupeol was shown to
inhibit protein kinase [17]. Recent studies
indicated that lupeol can significantly
decrease eosinophil infiltration and the
production of inflammatory cytokines such
as interleukin 4, 5, 6, and 13 (IL-4, IL-5, IL-6,
IL-13) by Th2 (T-helper type 2) cells. Therefore,
the presence of lupeol in silkworm excreta
supports its traditional anti-inflammatory use.
3.4 Safety Assessment of Silkworm
Excreta by Cytotoxicity Assay
Silkworm excreta and VLCFAs have
been used as food products and in dietary
supplements, respectively. It is thus important
to consider the safety of silkworm excreta
for consumption. In this study, both the crude
extract and 1-tritriacontanol were preliminary
tested for their cytotoxicity in both cancer cells
Chiang Mai J. Sci. 2014; 41(1)
and vero cells. Different types of cancer cells
were selected to represent different tissues,
such as those of the oral cavity, breast, and
lung. Vero cells were used to represent a
normal cell group. It was found that
%inhibition of both the crude extract and
1-tritriacontanol against all tested cancer cells
was less than 50%, which was interpreted as
inactive. In addition, %cell growth reported
in the vero cells assay was over 50%
indicating non-cytotoxic activity. The results
were interpreted by National Center for
Genetic Engineering and Biotechnology,
Thailand. The criterion for cytotoxicity
interpretation has been followed previous
studies [18]. These results suggested that the
crude extract and 1-tritriacontanol are not
toxic to those cell lines tested at the
concentration used.
Cytotoxicity has been known for short
chain alcohols such as alcohol while data
for the cytotoxic activities of long chain
alcohols or fatty alcohols are very limited.
In one study, crude fatty alcohols at
different concentrations were tested for
their cytotoxicity in a human cancer cell;
no toxicity was found [19]. However,
cytotoxicity has been found when fatty
alcohols are bound to sugar moieties to
form fatty alcohol glycosides. For example,
cupanioside, which is a long chain fatty
alcohol (hexadecyl) glycoside, showed in vitro
cytotoxicity against several cell lines
including HepG2, PC-3, Hs578T, and
MDA-MB-231 cells [19]. The cytotoxic
activity of fatty alcohol glycosides may be
due to their surfactant nature as surfactants
can interact with cell membranes, denature
enzymes, or bind to DNA or other cellular
substances, resulting in cell damage [20].
Concerning the toxicity of fatty alcohol
glycosides, it is worth noting that
contamination of fatty alcohol glycosides
during extraction and isolation of fatty
Chiang Mai J. Sci. 2014; 41(1)
alcohols may have occurred. Therefore,
thorough elimination of sugar moieties
from fatty alcohol glycosides is highly
recommended.
103
-HR). S.V. was a recipient of the Development
of New Faculty Staff grant from
Chulalongkorn University.
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3. CONCLUSIONS
This study was aimed at identifying
bioactive compounds from silkworm
excreta. Three compounds - 1-tritriacontanol,
lupeol, and β-sitosterol - were isolated. Of
these, 1-tritriacontanol and lupeol have not
previously been found in silkworm excreta.
It is likely that lupeol and β-sitosterol are
derived from the mulberry leaves on which
the silkworm fed and are excreted in an
unchanged form while 1-tritriacontanol is
likely synthesized in the silkworm intestine.
Identification of lupeol and β-sitosterol
supports the traditional medicinal use of
silkworm excreta in treating inflammation.
In addition, the cytotoxicity of the crude
extract and 1-tritriacontanol were tested and
found to be non-toxic to all cell types tested
in this study. According to the lipid profile
and fatty alcohols reported, silkworm excreta
are rich in liposoluble compounds particularly
fatty alcohols and fatty acids. Moreover,
silkworm excreta are very cheap and easily
harvested because they are inevitably and
abundantly produced as a major waste
product of sericulture. Therefore, the results
suggest that silkworm excreta would be a
good source of VLCFAs and could be used
for further studies to determine whether
silkworm excreta are a suitable commercial
source for VLFCAs. This would increase the
value of an otherwise useless by-product of
industrial sericulture and raise the income of
local farmers.
ACKNOWLEDGEMENTS
This research was financially supported
by Ratchadapiseksomphot Endowment Fund
of Chulalongkorn University (RES560530157
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