See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/225806168

Production of fermented needle extracts from red pine and their functional
characterization

Article  in  Biotechnology and Bioprocess Engineering · April 2008

DOI: 10.1007/s12257-008-0006-7

CITATIONS READS
10 378

6 authors, including:

Indeok Hwang Giri Raj Tripathi

Bunongseed Co. Ltd Tribhuvan University, Kirtipur, Kathmandu
29 PUBLICATIONS   292 CITATIONS    6 PUBLICATIONS   38 CITATIONS   

SEE PROFILE SEE PROFILE

Hyeonsook Cheong
Chosun University
60 PUBLICATIONS   1,361 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Molecular breeding of crop plants for secondary metabolites enrichment View project

All content following this page was uploaded by Giri Raj Tripathi on 03 December 2015.

The user has requested enhancement of the downloaded file.

Biotechnology and Bioprocess Engineering 2008, 13: 256-261
DOI/10.1007/s12257-008-0006-7

Production of Fermented Needle Extracts

from Red Pine and Their Functional
Characterization
=
d~óçìåÖ=m~êâI=aáääá=mK=m~ìÇó~äI=fåÇÉçâ=eï~åÖI=dáêá=oK=qêáé~íÜáI=vçìåÖâá=v~åÖI=~åÇ=
eóÉçåëççâ=`ÜÉçåÖG=
Department of Biotechnology and BK21 Research Team for Protein Activity Control, Chosun University, Gwangju, 501-759,
Korea

^Äëíê~Åí= Pine needle (máåìë=ÇÉåëáÑäçê~ Sieb. et Zucc.) extract is being used for millennia in treatment of multiple ailments in Asia Pa-
cific region. The extract is a composite mixture of a number of organic compounds. Terpenoid is one of such complex com-
pounds that exhibit useful effects. To remove harmful substances with improving functionality of the extract, people adopt
traditional spontaneous fermentation followed by aging. To assess the fermentation induced effectiveness we analyzed
composition of the volatile terpenoid in fresh to the 7 years old self-fermented extracts along with their anti-bacterial and anti-
oxidant properties. Simultaneously, we also analyzed the effect of remarkably changed 1,8-cineole and camphor. SFPEs
(self-fermented pine needle extracts), 1,8-cineole, and camphor inhibited growth of bacterial strains like p~äãçåÉää~ íóéÜáJ
ãìêáìã, pí~éÜóäçÅçÅÅìë=~ìêÉìë, bëÅÜÉêáÅÜá~=Åçäá, mëÉìÇçãçå~ë=~ÉêìÖáåçë~, and _~Åáääìë=ëìÄíáäáë. Comparative studies be-
tween pine needle extracts, 1,8-cineole and camphor showed that the self-fermented pine needle extracts retard the bacte-
rial growth effectively and have relatively higher antioxidant activities. PE (pine needle extract) and SFPEs showed nearly
95% DPPH radical scavenging activities, 1,8-cineole 84% while camphor showed no scavenging activities. The results
showed that self-fermentation and aging improves the effects of the extract therefore it could be a good source for nutraceu-
tical product. © KSBB

hÉóïçêÇëW=éáåÉ=åÉÉÇäÉ=Éñíê~ÅíëI=ëÉäÑJÑÉêãÉåí~íáçåI=íÉêéÉåçáÇëI=~åíáçñáÇ~åíI=~åíáÄ~ÅíÉêá~ä=
=
=
=
=
fkqolar`qflk=
Pinus densiflora is an evergreen needle-leafed tree indige-
nous to Asia. Biological activity of pine needle is the essence
for traditional medicine, which uses the pharmacological
efficacy of natural compounds present in pine needle for
treating human diseases. Red pine needles have long been
used as a nourishing tonic drug in Korean traditional medi-
cines and are frequently used to brew tea in Korea [1]. Fur-
thermore, pine needles are used in preparation of teas and
extracts. In connection with this, evidence has supported the
role that antioxidants, including several compounds, play in
the prevention of anti-aging and several chronic diseases
such as cardiovascular disease, cancer, diabetes, and anti-
hypertension [2]. Especially pine needle and bark are abun-
dant in terpenoids. Pine needles contain an essential oil
G`çêêÉëéçåÇáåÖ=~ìíÜçê=
Tel: +82-62-230-6667/7991 Fax: +82-62-224-6678
e-mail: hscheong@chosun.ac.kr
(0.3~1.3%), which contains α-pinene, β-pinene, camphene,
phellandrene, limonene, borneol (6.8%), and bornyl acetate
(3.8%) [3]. The essential oil of pine needles has found in
wide commercial use [4].
Terpenoids of different sizes and composition are found in
all classes of living things, the largest group of natural prod-
ucts, and are biologically active. Terpenoids and other nutri-
ent compounds of plants have been recognizes as potential
factors that can be useful to improve human health through
their antioxidant activities [5-7].
Plant terpenoids are aroma compounds that contribute
smell of eucalyptus, the flavors of cinnamon, cloves and
ginger, and the color of yellow flowers. They may exist as
vitamins, hormones, flavors, and biopolymer. They play
crucial roles in traditional herbal remedies and are under
investigation for their antibacterial and other pharmaceutical
effects. They are also employed for medical purposes. Anti-
malarial drug artemisinin and the anticancer drug paclitaxel
are among such terpenoids with established medical applica-
tions. Artemisinin is a drug used to treat multi-drug resistant

strains of falcioarum malaria. The compound (a sesquiter-
pene lactone) isolated from the shrub Artemisia annua is
long being used in traditional Chinese medicine [8]. And
paclitaxel is a mitotic inhibitor used in cancer (used to treat
patients with lung, ovarian, breast cancer, head, and neck
canner) chemotherapy [9]. In addition, presumably large
number of efficacious terpenoids is yet to be discovered.
Volatile components extracted using SDE method was iden-
tified 41 different compounds from the red pine needles. It is
also revealed that 8 or 10% (v/v) the extracts completely
inhibited the growth of Bacillus cereus, Salmonella typhi-
murium, and Staphylococcus aureus [3].
In this study, we produce first the self fermented pine nee-
dle extracts and determine their functional compounds or
roles with respect to antioxidant or antimicrobial effects.
This study attempts to reveal the trend of terpenoid com-
pounds dynamics in pine needle extracts from fresh one to 7
years fermented extract.
j^qbof^ip=^ka=jbqelap=
mä~åí=j~íÉêá~ä=
Fresh pine needles were harvested from Korean red pine
trees (Pinus densiflora Sieb. et Zucc.) in Gokseong, Jeol-
lanam-Do, Korea.
mêÉé~ê~íáçå=çÑ=máåÉ=kÉÉÇäÉ=bñíê~Åíë=
Pine needles (20 kg) were cleaned with tap water,
soaked with 5% charcoal in water, and homogenized with
mixer designed in our lab. The sample was incubated for 3
h at 4°C and the supernatant was recovered. The super-
natant was kept at 4°C for assays. Pine needle extract (PE)
was kept for three or seven years to induce emergence of
microorganisms, resulting in spontaneous fermentation in
extracts. The spontaneously fermented pine needle extracts
(SFPEs) and PE were detected their antioxidant and antim-
icrobial effects.
máåÉ=kÉÉÇäÉ=bñíê~Åíë=^å~äóëáë=Äó=d~ë=`Üêçã~íçÖê~éÜóJ=
j~ëë=péÉÅíêçãÉíêó=
Terpenoid compounds from pine needle extracts (PE,
SFPE 3, or SFPE 7) were analyzed by gas chromatogra-
phy-mass spectrometry (GC-MS) using HP-5 fusion silica
capillary column (0.25 mm × 30 cm × 0.25 μm) (Hewlett
Packard HP-5973 MSD and HP-6890, Agilent/Hewlett
Packard, USA). The PE and SFPEs were diluted 10 times
with distilled water and filtered using 0.45 μM nitrocellu-
lose membrane for injection containing the 25 mL frit
sparger, gas-tight syringes, purge and trap concentrator
(Tekmar 3000). The GC-MS conditions were following;
purge for 11 min, dry purge for 2 min, preheat at 175°C,
desorb at 180°C for 4 min, and trap bake at 180°C for 7
min. The carrier gas was 1 mL of helium gas per minute
with the speed of 36 cm/sec.
Biotechnol. Bioprocess Eng. ORT=
^åíáçñáÇ~åí=^ëë~ó=
Since camphor and 1,8-cineole were revealed as major ter-
pernoids from GC-MS analysis of pine needle extracts, they
were used for standard terpernoids in order to compare
functions of pine needle extracts. The radical-scavenging
activities of pine needle extracts, camphor, and 1,8-cineole
were evaluated by using the DPPH radical scavenging assay.
DPPH (150 μM) was dissolved in 50 mL of methanol. Sam-
ples were dissolved in methanol. Pine needle extracts were
diluted (1/100, w/v) in distilled water and assayed for their
antioxidant activity. The 1,8-cineole and camphor were pre-
pared in 0.15, 1.5, and 7.5 mg concentration per 0.1 mL. PE
or SFPEs (50 μg) were used for analyzing DPPH radical
scavenging activity. The mixture containing DPPH solution
and sample was incubated for 30 min at room temperature.
The absorbance was read against a blank at 517 nm. The
results were expressed as percentage of reduction of the ini-
tial DPPH adsorption by test samples.
^åíáÄ~ÅíÉêá~ä=^ëë~ó=
Pine needle extracts (PE, SFPE 3, or SFPE 7), camphor,
and 1,8-cineole were compared their antibacterial effects
against five bacterial strains. They are S. typhimurium (KCTC
1925), S. aureus (ATCC9341), Escherichia coli (KTCC1923),
Pseudomonas aeruginosa (ATCC15692), and Bacillus sub-
tilis (ATCC6633). Bacteria were cultured overnight at 37°C
in nutrient media (bacto beef extract 3 g, bacto peptone 5 g).
Antibacterial activities of the samples were detected by pa-
per disc method. Bacteria were grown for 16 h and its sus-
pension (100 μL) was spread on nutrient agar. Paper disks (6
mm diameter) were placed on the plate. Amount of extracts
PE or SFPE (200 μg/μL) and Camphor (150 μg/μL) was
dissolved in water. From the solution 20 μL was loaded on
the center of the paper disk and incubated at 37°C for 24 h.
The 1,8-cineole was used in final concentrations of 0.15, 1.5,
15, 75, and 150 μg/μL. Antibacterial activities of samples
were evaluated by measuring inhibition zone against the
each strain.
obpriqp=^ka=afp`rppflk=
qÉêéÉåçáÇë= nì~åíáÑáÅ~íáçå=áå=máåÉ= kÉÉÇäÉ= bñíê~Åíë=Äó=
d`Jjp=^å~äóëáë=
Volatile terpenoids in pine needle extracts (PE, SFPE 3, or
SFPE 7) have been identified and quantified by GC-MS
analysis as shown in Table 1. GC-MS analysis revealed that
α-pinene and 3-pentanone in PE were contained 9 and 11%,
respectively. Other minor compounds were identified as β-
myrcene, β-phellandrene, camphene, 3-carene, or eucalyptol.
Since pine tree needle retained α-pinene, β-phellandrene,
camphene, eucalyptol, and camphor as major volatile terpe-
noids [10,11]. Here, high amount of styrene in extract was
resulted from melted traces of plastic storage bottle. Interest-
ingly, camphor was not detected from the PE, however it
ORU=

q~ÄäÉ=NK= Volatile terpenoid compounds and essential oils in PE and purposed compound widely used in pharmaceutical products
SFPEs.= Trend of compounds dynamics in SFPEs. Inter- such as nasal decongestant, anticough agent, aromatheraphy,
change in compounds occurs and finally increases the to- dentistry, and in cosmetics [12,13]. Camphor is also terpe-
tal amount of terpenoids (especially 1,8-cineole and cam- noid, white or lucid solid with a strong, aromatic scent.
phor) due internal reactions during spontaneous fermenta- Camphor uses include as a moth repellent, as an antimicro-
tion. 1,8-Cineole and camphor concentration increased in bial substance, in embalming, in medicine [14]. Various ex-
during SFPE 3. Total concentration shows continuous in- periment tries showing the possible trend of some com-
creasing trend during spontaneous fermentation. pounds dynamics in fermented extracts. The aroma of wine
Compounds PE SFPE 3 SFPE 7
is secondary product formed during the wine fermentation
(fermentative aromas) and aging [15]. The aroma complexity
Alpha-pinene 9.303 increases during fermentation by the synthesis of important
Beta myrcene 0.536 volatile compounds [16]. SFPEs were assessed their func-
Beta phellandrene 1.370 tional properties with respect to antioxidant or antimicrobial
Camphene 2.230 effect.
Delta 3-carene 0.630
Antioxidant Properties of Pine Needle Extracts, 1,8-
Styrene 66.321
Cineole, and Camphor
3-Pentanone 11.707 7.439
Tolune 7.586 7.175 7.843 DPPH radical scavenging activity of pine extracts, cam-
1,8-Cineole/Eucalyptol 0.334 1.816 2.003 phor, and 1,8-cineole was also assessed (Fig. 3). 1,8-Cineole
2-Methyl-1-butanol 2.521 3.613
scavenged 46, 82, and 84% radicals from 0.15, 1.5, and 7.5
mg/100 μL) concentrations, respectively, and it was dose
3-Methyl-1-butanol 5.050 4.815
dependant (Fig. 3A), however camphor showed no scaveng-
5-Isopyrenyl-2-2-methyl-2-vinyltetrahydrofuran 4.021 7.555 ing activities. PE, SFPE 3, or SFPE 7 (50 μg) showed 95.4,
Bicyclo[2,2,1]heptan-2-ol 38.645 17.962 95.3, or 94.7% DPPH radicals scavenging activities, respec-
Camphor 6.888 24.195 tively (Fig. 3B). Lower concentration was found more effec-
Herboxide second isomer 9.139 10.906 tive than pure synthetic 1,8-cineole and camphor. As the
extract is a composite mixture of the several organic com-
Hexanoic acid, ethyl ester 1.359 1.193
pounds that might show higher anti-oxidant properties than
Isocineole 15.947 19.915 the terpenoid standard (1,8-cineole and camphor). Damage
Total terpenoic compounds (100%) 100 .100 100 by oxygen free radicals is known to be one of the mecha-
nisms of chronic disorders and ageing. Oxygen free radicals
are originated ubiquitously in aerobic metabolism, and are
was rapidly increased from 6.8% at the SFPE 3 to 24.2% at generally removed by antioxidant such as superoxide dismu-
SFPE 7. Eucalyptol was gradually increased with 0.3, 1.8, or tase, glutathione peroxidase, metal-binding proteins, vitamin
2.0% at PE, SFPE 3, or SFPE 7, respectively. C, vitamin E, beta carotene, uric acid, bilirubin, albumin,
Self fermentation did lead to produce the new compounds DNA repair enzyme, and methionine sulphoxide reductase
including methyl-butanol, vinyltetrahydrofuran, bicylco- repair [17,18]. Various reports try showing the antioxidant
heptanol, camphor, herboxide isomer, hexannoic acid, or effect in food. Red wine and green tea contains high level of
isocineole. Bicylco-heptanol, camphor, herboxide isomer, or antioxidant [19]. Self-fermented pine needle extract contains
isocineole were appeared in above 7% concentration as ma- several essential oils and terpenoids including 1,8-cineole
jor compounds in SFPE 3 or SFPE 7. and would have shown higher antioxidant activities than 1,8-
Both camphor and 1,8-cineole were known to be biologi- cineole.
cally active and volatile compounds as a common carbon
skeletal structure (Figs. 2A~2C). 5-Isopyrenyl-2-2-methyl-2- Antibacterial Properties of Pine Needle Extracts, 1,8-
vinyltetrahydrofuran (4.021 to 7.555%), herboxide second Cineole, and Camphor
isomer (9.139 to 10.906%), and isocineole (15.947 to
19.915%) are the characteristic changes occurred in the PE Pine needle extracts (PE, SFPE 3, and SFPE 7), 1,8-
to SFPEs. Those results revealed that the compositions of cineole, and camphor were studied for their antibacterial
terpenoids were changed rapidly during spontaneous fermen- functions. The experiment showed that pine needle extracts,
tation process (Table 1). Volatile compounds of self- 1,8-cinoele, and camphor can retard growth of bacteria. The
fermented pine needle extracts were detected using GC-MS. 1,8-cineole and camphor were so sensitive to S. typhimurium,
Especially, peak a (11.03 min) corresponding to 1,8-cineole, S. aureus, E. coli, P. aeruginosa and B. subtilis. The 1,8-
peak b (13.02 min) corresponding to camphor (Fig. 1). 1,8- cineole application (15 μg/μL) resulted in growth inhibition
Cineole is natural organic compound and called as eucalyp- of E. coli and B. subtilis within 6 and 7 mm distance. Higher
tol, eucalyptole, limonene oxide, cajeputol, 1,8-epoxy-p- amount of 1,8-cineole (75 μg/μL) resulted in inhibition of
menthane, 1,3,3-trimethyl-2-oxabicyclo[2,2,2]octane, 1,8- individual bacteria growth of S. typhimurium, S. aureus, E.
oxido-p-menthane, cineol, or cineole [8]. It is a multi- coli, P. aeruginosa, or B. subtilis within 20, 12, 20, 15, or 20
 Biotechnol. Bioprocess Eng. ORV=

^=

_=

`=

cáÖK=NK= Gas chromatography (GC-MS) profile of self-fermented pine needle extract.=Volatile compounds of self-ferm-ented pine needle
extract detected using GC/MS. Peak a (11.03 min) corresponding to 1,8-cineole, peak b (13.02 min) corresponding to camphor.
The time and mass spectra were compared with that of the standard. A, PE; B, SFPE 3; C, SFPE 7.=

^= = = = = = = = = = = = = = = = _= = = = = = = = = = = = = `= P. aeruginosa (8 mm), and B. subtilis (12 mm) have been

cáÖK=OK= Structures of camphor and 1,8-cineole. Molecular struc-
tures of camphor (A), 1,8-cineole (B and C).
mm distance, respectively. The 1,8-cineole application (150
μg/μL) resulted in growth inhibition S. typhimurium, S.
aureus, E. coli, P. aeruginosa, or B. subtilis within 25, 16,
25, 20, or 27 mm distance, respectively (Fig. 4). However,
treatment of 20 μL (150 μg/μL) of camphor on S. typhi-
murium, B. subtilis, and P. aeruginosa showed equal (8 mm)
inhibition to all the tested strains. Treatment of 4 mg SFPEs
showed highest inhibition to growth of all strains tested. S.
typhimurium (12 mm), S. aureus (10 mm), E. coli (10 mm),
inhibited by SFPE 7. Effect of SFPE 3 was lower than the
SFPE 7, however PE showed no effect to all strains (Table 2,
Figs. 4 and 5). Tested bacteria were highly susceptible to
SFPEs, however showed low susceptibility to 1,8-cineole
and camphor. These properties of pine needle extract might
have gained due to synergistic effect of the compounds
changed during self-fermentation process [20]. The food and
cosmetic industry at present is facing an enormous problem
from user for using chemical preservatives to prevent the
growth of microorganisms [21,22]. Antibacterial properties
of the terpenoids (essential oils) and a variety of extracts
from many plants have recently been of great interest in both
study, the food and cosmetic industry, because their potential
use as organic additives emerged from a increasing trend to
replace synthetic preservatives with natural ones. It is known
that red pine needles have long been valuated for their medi-
cal effects and have been used in popular medicine for the
treatment of hepatopsis, various neurological disorders, and
OSM=

^=

cáÖK=QK= Antibacterial effect of 1,8-cineole standard.=Antimicrobial

effect of 1,8-cineole was analyzed by paper disc method.
_= Figure shows the effect of 1,8-cineole to different bacte-
rial strains on paper disc. The 1,8-cineole suppressed the
bacterial growth in dose dependent manner. DMSO was
used as negative control.

cáÖK=PK= DPPH radical scavenging activity of PE, SFPE 3, SFPE 7,

1,8-cineole, and camphor.= DPPH radical scavenging
activities of 1,8-cineole (A), PE, SFPE 3, SFPE 7,
camphor, and 1,8-cineole (B). DMSO was used as nega-
tive control.

arteriosclerosis [4]. Also the volatile components of pine nee-

dles inhibit growth of pathogenic bacteria [3]. Therefore, a
new approach to prevent the proliferation of microorganism
might safeguard against biological invasion in various healthy
food products from red pine extracts, however it needs detail
assessments in components improvement mechanism.
CONCLUSION
cáÖK=RK= Antibacterial effects of SFPE 7, camphor, and 1,8-
cineole.=Bacterial strains were susceptible to 1,8-cineole
and SFPE, but were not to camphor. Amount of extracts
PE or SFPE 7 (200 µg/µL), camphor (150 µg/µL), and
1,8-cineole (150 µg/µL) was dissolved in water. From the
solution 20 µL was loaded on the center of the paper
disk.

Various aspects of analyses of self-fermented pine needle

extracts reveal that they contain several useful compounds q~ÄäÉK=OK=Antimicrobial activities of PE, SFPEs, camphor, and
that are additive as nourishing agents. Essential oils and ter- 1,8-cineole was analyzed by paper disc method
penoid compounds have been found in self-fermented pine
needle extracts. Terpenoid compounds (1,8-cineole and Bacterial strains
Pine extracts (4 mg) Compounds (3 mg)
camphor) from self-fermented pine needle extract possess PE SFPE 3 SFPE 7 1,8-Cineole Camphor
antibacterial and antioxidant properties where effectiveness pK=íóéÜáãìêáìã 08 12 25 8
increases with increases at treatment concentration. Com- pK=~ìêÉìë 09 10 16
parative studies between pine needle extracts, 1,8-cineole
bK=Åçäá 10 25
and camphor showed that SFPEs and 1,8-cineole have anti-
bacterial and antioxidant properties. Tested bacteria were mK=~ÉêìÖáåçë~= 08 20 8
highly susceptible to SFPEs, however showed low suscepti- _K=ëìÄíáäáë 10 12 27 8
bility to 1,8-cineole and camphor. Similarly, DPPH radical PE, SFPE 3, and SFPE 7 4 mg in final volume 40 µL; camphor and 1,8-cineole 3
scavenging activities of pine needle extract was higher than mg in final volume 20=µL: effect was measured in inhibition distance in millimeter.

1,8-cineole while no scavenging effect was shown by cam-
phor. These properties of pine needle extract might have
gained due to synergistic effect of the compounds changed
during self-fermentation process.
^ÅâåçïäÉÇÖÉãÉåíë This Research was supported by the
“Program for the Training of Graduate Students in Regional Inno-
vation”, which was conducted by the Ministry of Commerce, Indus-
try and Energy of the Korea Government. This study was also par-
tially supported Youngki Yang by research funds from Chosun
University, 2003.
Received January 14, 2008; accepted April 11, 2008
obcbobk`bp=
1. Jung, M. J., H. Y. Chung, J. H. Choi, and J. S. Choi
(2003) Antioxidant principles from the needles of red
pine, Pinus densiflora. Phytother. Res. 17: 1064-1068.
2. Yen, G. C., P. D. Duh, D. W. Huang, C. L. Hsu, and T.
Y. Fu (2008) Protective effect of pine (Pinus morrisoni-
cola Hay.) needle on LDL oxidation and its anti-
inflammatory action by modulation of iNOS and COX-2
expression in LPS-stimulated RAW 264.7 macrophages.
Food Chem. Toxicol. 46: 175-185.
3. Kim, Y. S. and D. H. Shin (2005) Volatile components
and antibacterial effects of pine needle (Pinus densiflora
S. and Z.) extracts. Food Microbiol. 22: 37-45.
4. Lee, J. G., C. G. Lee, J. J. Kwag, A. J. Buglass, and G.
H. Lee (2005) Determination of optimum conditions for
the analysis of volatile components in pine needles by
double-shot pyrolysis-gas chromatography-mass spec-
trometry. J. Chromatogr. A 1089: 227-234.
5. Chang, S. F., C. L. Hsieh, and G. C. Yen (2008) The
protective effect of Opuntia dillenii Haw fruit against
low-density lipoprotein peroxidation and its active com-
pounds. Food Chem. 106: 569-575.
6. Lee, Y. J., T. Y. Kim, and H. W. Chung (2001) Protec-
tive effects of Ginkgo biloba leaf extract (GBE) against
1,2,4-benzenetriol induced toxicity in vitro. Kor. J. Env.
Hlth. Soc. 1: 124-130.
7. Pietri, S., E. Maurelli, K. Drieu, and M. Culcasi (1997)
Cardioprotective and anti-oxidant effects of the terpe-
noid constituents of Ginkgo biloba extract (EGb 761). J.
Mol. Cell Cardiol. 29: 733-742.
View publication stats
Biotechnol. Bioprocess Eng. OSN=
8. http://en.wikipedia.org/wiki/Eucalyptol.
9. http://en.wikipedia.org/wiki/Paclitaxel.
10. Canini, A., D. Alesiani, G. D’Arcangelo, and P. Tagliat-
esta (2007) Gas chromatography-mass spectrometry
analysis of phenolic compounds from Carica papaya L.
leaf. J. Food Compost. Anal. 20: 584-590.
11. Byun, H. G., T. K. Eom, W. K. Jung, and S. K. Kim
(2007) Lipase catalyzed production of monoacylglyc-
erols by the esterification of fish oil fatty acids with
glycerol. Biotechnol. Bioprocess Eng. 12: 491-496.
12. Madyastha, K. M. and A. Chadha (1986) Metabolism of
1,8-cineole in rat: its effects on liver and lung micro-
somal cytochrome P-450 systems. Bull. Environ. Con-
tam. Toxicol. 37: 759-766.
13. Soares, M. C. M. S., C. E. N. Damiani, C. M. Moreira, I.
Stefanon, and D. V. Vassallo (2005) Eucalyptol, an es-
sential oil, reduces contractile activity in rat cardiac
muscle. Braz. J. Med. Biol. Res. 38: 453-461.
14. http://en.wikipedia.org/wiki/Camphor.
15. Swiegers, J. H. and I. S. Pretorius (2005) Yeast modula-
tion of wine flavor. Adv. Appl. Microbiol. 57: 131-175.
16. Swiegers, J. H., E. J. Bartowsky, P. A. Henschke, and I.
S. Pretorius (2005) Yeast and bacterial modulation of
wine aroma and flavour. Aust. J. Grape Wine Res. 11:
139-173.
17. Sung, H., J. Nah, S. Chun, H. Park, S. E. Yang, and W.
K. Min (2000) In vivo antioxidant effect of green tea.
Eur. J. Clin. Nutr. 54: 527-529.
18. Seo, Y., H. J. Lee, K. E. Park, Y. A. Kim, J. W. Ahn, J.
S. Yoo, and B. J. Lee (2004) Peroxynitrite-scavenging
constituents from the brown alga Sargassum thunbergii.
Biotechnol. Bioprocess Eng. 9: 212-216.
19. Maxwell, S., A. Cruickshank, and G. Thorpe (1994) Red
wine and antioxidant activity in serum. Lancet 344: 193-
194.
20. Park, G. Y., H. X. Li, I. Hwang, and H. Cheong (2006)
The functional effects of fermented pine needle extract.
Kor. J. Biotechnol. Bioeng. 21: 376-383.
21. Deba, F., T. D. Xuan, M. Yasuda, and S. Tawata (2008)
Chemical composition and antioxidant, antibacterial and
anti fungal activities of the essential oils from Bidens pi-
losa Linn. var. Radiata. Food Control 19: 346-352.
22. Kim, I. H., D. G. Lee, S. H. Lee, J. M. Ha, B. J. Ha, S. K.
Kim, and J. H. Lee (2007) Antibacterial activity of Ulva
lactuca against methicillin-resistant Staphylococcus
aureus (MRSA). Biotechnol. Bioprocess Eng. 12: 579-
582.