Separation and Purification Technology 146 (2015) 1–7

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Separation and Purification Technology

journal homepage: www.elsevier.com/locate/seppur

Simultaneous extraction of nicotine and solanesol from waste tobacco

materials by the column chromatographic extraction method and their
separation and purification
Ri-Sheng Hu a,b, Juan Wang b, Hui Li b, He Ni b, Ya-Fei Chen b, Ya-Wen Zhang b,c, Shi-Peng Xiang a,
Hai-Hang Li b,⇑
a
South Central Agriculture Experimental Station of China Tobacco, Changsha, Hunan 410128, China
b
Guangdong Provincial Key Lab of Biotechnology for Plant Development, College of Life Sciences, and Research and Development Center for Rare Animals, South China Normal
University, Guangzhou 510631, China
c
Guangzhou Huichuan Medical Technology Company, 211 Jinfu Building, 90 Qifu Road, Baiyun District, Guangzhou 510410, China

a r t i c l e i n f o a b s t r a c t

Article history: The tobacco (Nicotiana tabacum L.) cultivation and cigarettes manufacture industries discard huge
Received 30 January 2015 amount of waste tobacco materials that have strong nicotine smell and contaminate the environment.
Received in revised form 15 March 2015 A high-efficient procedure was developed for simultaneously extracting nicotine and solanesol from
Accepted 17 March 2015
the waste tobacco leaf vein through the column-chromatographic extraction (CCE), followed by
Available online 23 March 2015
automatically separating and simply purifying them. Dried material powder was loaded into columns
and eluted with the optimal extraction solvent of petroleum ether (PE):95% alkali ethanol (4:6).
Keywords:
Greater than 96% extraction efficiency for both nicotine and solanesol was obtained with a 2-fold excess
Waste tobacco material
Column chromatographic extraction
solvent of the material (v/w) through a cyclic CCE procedure in small-scale and scaled-up experiments.
Purification The extraction solution was separated into an ethanol-aqueous phase containing 98% nicotine and an
Nicotine ether phase containing 96% solanesol at pH 2.0. The ethanol-aqueous phase was vacuum-concentrated
Solanesol to aqueous, and 99% purity nicotine was obtained by ether fractionation of the aqueous at pH 10.0.
Solanesol in the ether phase was purified to 93.1% by one time silica gel column chromatography. All pro-
cesses were completed at room temperature and all solvents used were completely recovered for reuse.
This work provides an extensively simplified procedure to economically utilize the tobacco wastes.
Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction Nicotine is widely used in fine chemical, pharmaceutical and

Tobacco (Nicotiana tabacum L.) is one of the most widely
planted economic crops. China is the largest tobacco producer
and consumer in the world with an annual tobacco production
reached 400–500 million tons. In the tobacco cultivation and cigar-
ette manufacture industries, more than 200 million tons of waste
tobacco materials are produced annually. The wasters, including
stem, leaf vein and roots of tobacco plants, low grade and defective
tobacco leaves, have strong smell and cause serious environmental
contamination [1]. The tobacco wastes contain high amount of
nicotine and solanesol and are valuable resources for the extrac-
tion of the two bioactive compounds [2]. After extraction of nico-
tine and solanesol, the plant residues can be used for multiple
purposes, such as fiberboards, pulps and organic fertilizers.
⇑ Corresponding author. Tel./fax: +86 20 8521 2630.
E-mail address: li_haihang@yahoo.com (H.-H. Li).
agriculture industries, and in the tobacco industry itself as an
essential cigarette additive. Nicotine is reported to improve the
health conditions of patients with diseases of dementia [3] and
schizophrenia [4], dopaminergic neurons and axons [5], skin mild
cognitive dysfunction [6], levodopa induced dyskinesia [7], and
to reduce the intake of harmful substances of smokers in nicotine
aided smoking cessation [8]. Nicotine has antimicrobial [2] and
insecticidal activities and has been used as a natural insecticide
with characteristics of easily degradable, non-toxic to human being
and no environmental pollution [9]. Solanesol has useful medicinal
properties and possesses anti-oxidant, anti-bacterial, anti-in-
flammation, and anti-ulcer activities [10]. Industrially, solanesol
is used by the pharmaceutical industry as an intermediate in the
synthesis of metabolically active quinones such as coenzyme Q10
and vitamin K analogues. The demand for solanesol continues to
escalate since coenzyme Q10 entered the market as a dietary sup-
plement [10].

http://dx.doi.org/10.1016/j.seppur.2015.03.016
1383-5866/Ó 2015 Elsevier B.V. All rights reserved.
2 R.-S. Hu et al. / Separation and Purification Technology 146 (2015) 1–7
Tobacco is the main natural source for the isolation of nicotine
and solanesol. Many protocols have been published for the extrac-
tion of nicotine and solanesol from tobacco leaves and wastes,
which includes heat reflux extraction [11], solid phase extraction
[12], ultrasound-assisted [11,13] or microwave-assisted extrac-
tions [14,15], and supercritical fluid extraction [16]. However,
these reports are mainly for the extraction of a single compound.
They need heating, or large volumes of organic solvents for the
extraction. The separation and purification of solanesol used more
than one time of chromatographic steps, including silica gel chro-
matography [17,18], Bio-Beads or Sephadex LH-20 gel chro-
matography [19], counter current chromatography [20], or their
combinations. These methods are not cost-effective for compre-
hensive utilizing the tobacco wastes.
We have reported a highly efficient column-chromatographic
extraction (CCE) method for the extraction of natural compounds
from biological materials [21–23]. In this work, we report a unique
procedure for simultaneously extracting nicotine and solanesol
and hydrolyzing bound forms or ester forms of nicotine and sola-
nesol using a minimum volume of the optimal solvent, followed
by automatic separation and simple purification to economically
produce highly-purified nicotine and solanesol using the waste
tobacco materials.
2. Materials and methods
2.1. Waste tobacco material and reagents
The waste tobacco material (leaf vein with a few broken leaves)
is a cigarette manufacture waste provided by Changsha Cigarette
Factory. The material was dried in a 45 °C oven for 4 h, ground
and passed through a 40-mesh sieve. Materials less than 40-mesh
were used for the experiments.
Standard compounds of nicotine and solanesol were purchased
from the National Institute for Food and Drug Control of China.
HPLC solvents were purchased from Burdick & Jackson Inc.
(Muskegon, MI, USA). Food-grade ethanol (95%) was used in all
extractions and purifications. Other analytical- or biochemical-
grade organic solvents and chemical reagents were purchased from
local suppliers.
2.2. Analysis of nicotine and solanesol by TLC and HPLC
Silica gel G TLC plates (10  10 mm, Merck Co., Germany) were
used for qualitative analysis of nicotine and solanesol. The devel-
oping solvents were chloroform:methanol (10:1, v/v) for nicotine
analysis, and 1,2-dichloroethane for solanesol analysis. All TLC
plates were stained in iodine vapor after developed.
A Shimadzu SPD-20A HPLC system (Shimadzu, Japan) with LC-
20AT UV detector and YMC-packed ODS column (250 mm  4.6
mm, 5 lm) was used for qualitative and quantitative analyzes of
nicotine and solanesol. Nicotine was analyzed using a mobile
phase of methanol:aqueous solution of 0.2% triethylamine (4:6,
v/v) at a flow rate of 0.8 mL/min and detected at 254 nm.
Solanesol was analyzed using a mobile phase of 100% methanol
at a flow rate of 0.8 mL/min and detected at 215 nm. All samples
for HPLC analysis were filtered through 0.45 lm membrane filters
before injection. Nicotine and solanesol in samples were identified
by the retention times and co-injection tests with their
corresponding standard compounds. Their amounts were deter-
mined by their peak areas based on the standard curves.
Fig. 1 shows the results of optimized HPLC analyzes of nicotine
(Fig. 1A and B) and solanesol (Fig 1C and D) of standard compounds
and the extracts from the waste tobacco material. The nicotine
content had good linear relationship with the peak area in the
range from 5 lg to 35 lg (Fig. 1E). Solanesol content had good lin-
ear relationship with the peak area in the range from 0.2 lg to
35 lg (Fig. 1F).
The analytic methods had high precision with RSDs (relative
standard derivation, n = 5) of 0.75% for nicotine and 0.42% for sola-
nesol. Repeatability tests showed that the RSDs (n = 5) were 1.01%
and 0.12%, with recovery rates of 99.81% ± 0.52% and 99.81% ±
0.59% for nicotine and solanesol, respectively. Nicotine (in 95%
ethanol) and solanesol (in PE) were stable within 24 h at room
temperature, and the RSDs of their amounts were 1.70% for nico-
tine and 0.55% for solanesol at 24 h.
2.3. Selection of extraction solvents
To select the extraction solvent that best dissolves nicotine and
solanesol in the waste tobacco material, 0.5 g of dried material was
added into 5 mL extraction solvents (petroleum ether or
PE:ethanol from 10:0, 8:2, 6:4, 4:6, 2:8 or 0:10) and incubated at
room temperature (30 °C water bath) for one hour with 20 rpm/
min shaking. After centrifugation at 5000g for 10 min, nicotine
and solanesol in the solutions were analyzed by HPLC.
2.4. Determination of minimum solvent volume and minimum
dissolution time
Dried material (0.5 g) was added into 5 mL extraction solvent
and incubated at room temperature (30 °C water bath) for different
hours (1–5 h) with 20 rpm shaking. The solution and tobacco resi-
due were then separated by centrifugation at 5000g for 10 min. The
solvent volume required for the tobacco material fully imbibed
was determined as the minimum volume (MV). MV is a basic vol-
ume unit in the extraction. The contents of nicotine and solanesol
in the solutions were determined by HPLC, and the minimum times
for the two compounds fully dissolved were determined.
2.5. Column-chromatographic extraction
CCE was performed in glass chromatographic columns [21–23].
For non-cyclic CCE, 8 g tobacco material was loaded into a column
with one MV solvent at a bed height to diameter ratio of 10:1 or
5:1, through the wet column preparation method. After remained
for 3 h until nicotine and solanesol dissolved, the column was
eluted with the extraction solvent at a flow rate of one MV/h.
The eluent was collected in fractions (one MV each) and the con-
tents of nicotine and solanesol in each fraction were analyzed by
HPLC.
For cyclic CCE, two MVs eluent was collected from the column,
the first MV was collected as the extraction solution, and the sec-
ond MV was used to extract the next batch material. The volume
of extraction solution was one MV.
2.6. Separation of nicotine and solanesol
The extraction solution was adjusted to pH 2.0 (with 1 mol HCl
for all pH adjustment) and separated into two phases. The ethanol
aqueous phase was washed with 1/4 volume PE, and the PE phase
was washed with 1/4 volume of 90% acidic ethanol (pH 2.0). The
nicotine aqueous solution was obtained after vacuum-concentra-
tion of the combined ethanol aqueous phase. Crude solanesol
was obtained after vacuum-concentration of the combined PE
phase.
2.7. Purification of nicotine
The nicotine aqueous solution was adjusted to pH 10.0 and frac-
tionated with PE for three times with equal volume for first time
 R.-S. Hu et al. / Separation and Purification Technology 146 (2015) 1–7
Fig. 1. HPLC analyzes of nicotine ((A) standard; (B) extracts) and solanesol ((C) standard; (D) extracts) and their quantitative curves ((E) nicotine; (F) solanesol).
and half volume for the second and third times. The combined PE
phase was washed once with 1/4 volume of alkaline water (pH
10.0). Purified nicotine was obtained after vacuum recovery of
the PE.
2.8. Purification of solanesol
The crude solanesol was purified by silica gel (200–300 mesh)
column chromatography. 1,2-Dichloroethane was used to prepare
the column, dissolve and load the sample onto the column and
elute the column. The eluent was collected in fractions each with
3 BVs (bed volumes of the column). All fractions were analyzed
by silica gel TLC and HPLC. Fractions with pure solanesol were
combined together and vacuum-concentrated.
All quantitative experiments were repeated at least three times,
and the results are the means of three independent experi-
ments ± standard error.
3. Results and discussion
3.1. Extraction solvent and conditions
Extraction of nicotine and solanesol was performed with the
CCE method using the PE–ethanol–water solvent system. The CCE
method includes two steps, dissolving and eluting the target com-
pounds from the plant material in chromatographic columns with
minimum volume solvent at room temperature [21–23]. As shown
in Fig. 2A and B, in a series of different ratios of PE–95% ethanol, the
extraction efficiency of both nicotine and solanesol increased with
the increase of ethanol proportion from 0% to 60%, and then
decreased when ethanol proportion is more than 60%. PE:95% etha-
nol at 4:6 was the best solvent for extracting nicotine and solanesol
at the same time. The minimum time for nicotine and solanesol to
be fully dissolved in the extraction solution was 3 h (Fig. 2C and D).
The MV of the extraction solvent for the tobacco material being
fully imbibed was determined to be 2.0-fold of the material
3
(v/w), which means 1.0 g material absorbed maximally 2 mL sol-
vent (data not shown in the figures).
It was reported that some of the nicotine and solanesol existed
in bound or ester form in the tobacco materials. Alkali or acid treat-
ment of the tobacco materials can release the bound form increase
the extraction rate of nicotine [24], and a saponification step can
hydrolyze solanesol esters and increase solanesol content after
extraction [14,25,26]. Simultaneous saponification of solanesol
esters and releasing bound form of nicotine in the extraction pro-
cess were tested by adding different amount (0.5%, 1%, 2% and 4%)
of NaOH in the 95% ethanol of extraction solvent. The results
showed that, comparing to the control (100%) without NaOH in
the extraction solvent, 0.5% NaOH did not have significant effect
on the extraction of nicotine and solanesol, the addition of 1.0%,
2.0% or 4.0% NaOH increased the contents of nicotine and solanesol
significantly. 2.0% NaOH showed the highest extraction for both
compounds, which increased the contents of nicotine and solane-
sol by 12% and 20%, respectively (Fig. 2E and F). After extraction,
the extraction solution was re-saponified with NaOH, and the con-
tents of both nicotine and solanesol did not increase (data not
shown), which indicated that the saponification of solanesol esters
and the releasing of bound nicotine were completed in the extrac-
tion step with alkali solvent. The extraction solvent was deter-
mined to be PE:95% alkali ethanol containing 2% NaOH at 4:6.
3.2. Column-chromatographic extraction of nicotine and solanesol
Based on the results above, nicotine and solanesol were
extracted from the waste tobacco material in small-scale (8 g)
and in scale-up (1.6 kg) experiments through the CCE procedure.
Dried tobacco material powder was loaded with 1.0 MV (or 2-fold,
v/w) extraction solvent into columns and eluted with the extrac-
tion solvent. Three MVs of eluent was collected and analyzed by
HPLC. In small-scale experiments, 99% nicotine in the tobacco
material was extracted in 3 MVs eluent, with 86%, 10% and 3% of
the total amount in the 1st, 2nd and 3rd MV eluent, respectively
4 R.-S. Hu et al. / Separation and Purification Technology 146 (2015) 1–7

Fig. 2. Effects of different factors on the extraction of nicotine and solanesol from the tobacco material. (A and B) different ratios of petroleum ether (PE) to 95% ethanol; (C
and D) different dissolving times with PE to 95% ethanol of 4:6; (E and F) the effect of different amounts of NaOH in the 95% ethanol of the solvent on the extraction of nicotine
and solanesol.

Fig. 3. Extraction efficiency of nicotine (A and C) and solanesol (B and D) from the tobacco material in small scale (A and B) and scale-up (C and D) experiments. Tobacco
material was extracted by CCE method with PE:95% ethanol (containing 2% NaOH) 4:6 in columns at height-to-diameter (H/D) of 5:1 (white bars) and 10:1 (black bars). 2.0C
represents cyclic extraction with 2.0-fold solvent of the dried tobacco material, others are for non-cyclic extraction.

(Fig. 3A); and 100% solanesol was extracted in 3 MVs eluent, with
97%, 2% and less than 1% of the total solanesol in the 1st, 2nd and
3rd MV eluent, respectively (Fig. 3B).
As most of the nicotine and solanesol were in the 1st MV eluent,
in order to reduce the use of solvent and the volume of the final
extraction solution, cyclic CCE was tested. In the cyclic CCE, only
the 1st MV eluent was collected as extraction solution, and the
2nd MVs eluent was used to extract the next material. Through
the cyclic CCE, 96% nicotine (the 1Cyl in Fig. 3A) and 99% solanesol
(the 1Cyl in Fig. 3B) were extracted from the material using 1 MV
or 2-fold extraction solvent. Two height-to-diameter (H/D) ratios
(5:1 and 10:1) of the column loaded were tested for the extraction
of both nicotine and solanesol, the H/D 10:1 showed 1–2% higher
extraction efficiency in the first MV eluent, but had the same
extraction efficiency when collected 2 or 3 MVs eluent
(Fig. 3A and B).
The experiments were scaled-up 200 times with 1.6 kg of dried
powder of the waste tobacco material in a 10 cm diameter column.
Similar to the small-scale experiments, 95% nicotine and 99% sola-
nesol were extracted with 1 MV of 2-fold extraction solvent
through the cyclic CCE (Fig. 3C and D).
3.3. Separation of nicotine and solanesol
The mixture of PE–95% ethanol is a uniform single phase sol-
vent, and separates into two phases when the water content in
 R.-S. Hu et al. / Separation and Purification Technology 146 (2015) 1–7 5

the ethanol is about 10% or more. As the water in the tobacco

material can be partially extracted which increased water content
in the solvent, the extraction solution was automatically separated
into an ethanol aqueous phase and a PE phase. As shown in Fig. 4,
most nicotine was found in the ethanol aqueous phase and most
solanesol was in the PE phase. The separation ratios were depen-
dent on the pH value of the solution. Nicotine and solanesol was
best separated at pH 2.0, with 98% nicotine in the ethanol aqueous
phase and 96% solanesol in the PE phase. The ethanol aqueous
phase was washed with 1/4 volume of PE, and the PE phase was
washed with 1/4 volume of 90% acidic ethanol (pH 2.0). The nico-
tine aqueous solution was obtained after vacuum-recovery of etha-
nol of the combined ethanol aqueous phase. Crude solanesol
(40.2%) was obtained after vacuum-recovery of PE of the combined
PE phase. The organic solvents of PE and ethanol used in the
extraction and separation steps can be clearly separated and vac-
uum-recovered for reuse.

3.4. Purification of nicotine

As shown in Fig. 5, nicotine was mainly in the water phase in

acidic condition (pH 6.0 or lower), and mainly in the PE phase in
alkali condition (pH 8.0 or higher) in the PE–water fractionation
system. At pH 10.0 or higher, 94% nicotine was found in the organic
phase. The nicotine aqueous solution obtained in Section 3.4 was
adjusted to alkali conditions and fractionated with PE for two
times, equal volume for the first and 1/4 volume for the second
time. The combined PE phase was washed with 1/4 volume of alka-
Fig. 5. Nicotine distribution in the petroleum ether phase (white bars) and aqueous
line water. Purified nicotine with 99.3% purity was obtained after phase (black bars) fractionated at different pH values (A) and HPLC analysis of
vacuum-recovering the PE. The overall recovery rate of nicotine purified nicotine (B).
from the extraction to purified product was 93.6%. PE is the only
organic solvent in this step, the vacuum-recovered PE is pure and
3.5. Purification of solanesol
can be reused directly.

It is difficult to purify solanesol to more than 90% purity with-

Fig. 4. Distribution of nicotine (A) and solanesol (B) in petroleum ether (white bars)
and ethanol aqueous (black bars) phases of the extraction.
out using the column chromatography. The crude solanesol (con-
taining 40.2% solanesol) was purified by a silica gel column
chromatography. A series of single solvent and mixtures of two
or three solvents were tested as the mobile phase, and the single
solvent 1,2-dichloroethane showed the best performance for sepa-
rating solanesol. The crude solanesol was dissolved, loaded onto
the column and eluted with 1,2-dichloroethane. TLC analysis indi-
cated that the eluent fractions from 13 to 19 contained purified
solanesol (Fig. 6A) and were combined together. HPLC analysis
(Fig. 6B) indicated that the purified product contained 93.1% sola-
nesol with a recovery rate of 87.4% in the column chromatographic
step. The overall recovery rate of solanesol was 82.6% in the extrac-
tion, separation and purification steps. As 1,2-dichloroethane was
the only solvent used in the whole chromatographic process, it
was easily recovered for reuse by vacuum-distillation of all eluted
fractions.
Most of the reports on the extraction of nicotine and solanesol
from tobacco materials are for single compound or for the two
compounds, but by two independent extraction steps [26].
Furthermore, the traditional solvent extraction methods used more
than 10-fold solvent [14,26] and extracted the materials for two or
more times [12,27] in microwave-assisted [14,15] or ultrasound-
assisted [11,26] maceration extractions, or extracted the materials
at high temperatures in refluxing extraction [12,27]. Our work pre-
sented here combined the extraction of nicotine and solanesol, the
saponification or hydrolysis of solanesol esters and the release of
bound form nicotine into one step using a carefully designed alkali
extraction solvent. The CCE method used simple columns as
extraction equipment and extracted both compounds by more
than 96% with only 1 MV or 2-fold solvent at room temperature.
The extraction solution was automatically separated into PE and
6 R.-S. Hu et al. / Separation and Purification Technology 146 (2015) 1–7
Fig. 6. TLC (A) and HPLC (C) analysis of solanesol from silica gel column separation (A) TLC analysis of different fractions eluted from silica gel column chromatogram, the TLC
plate was developed with 100% 1,2-dichloroethane. S, solanesol standard, O, original sample before column separation; (B) HPLC analysis of purified solanesol (C) in 1,2-
dichloroethane (B) by silica gel column chromatogram.
Fig. 7. Diagram of the extraction, separation and purification of nicotine and
solanesol from tobacco material.
ethanol-aqueous phases, which not only completely separated
solanesol and nicotine but also made the solvents be easily recov-
ered for reuse.
In the processes of purifying nicotine and solanesol, we avoided
using high-cost purification methods or more than one step of
chromatography, such as preparative HPLC, Bio-Beads chro-
matography, Sephadex LH-20 chromatography [19], HCCC [20,28]
and their combinations. In our procedure, nicotine was purified
by one-step solvent fractionation at pH 10. Solanesol was purified
by single-step silica gel column chromatography with 1,2-dichlor-
oethane as a mobile phase, which was also easily recovered for
reuse. The overall procedure was summarized in Fig. 7.
4. Conclusions
This work developed a highly efficient procedure for simultane-
ous extraction of nicotine and solanesol from the waste tobacco
material with minimum volume of solvent at room temperature,
followed by automatically separating and simply purifying them.
Dried tobacco material was loaded into a column and eluted with
the optimal extraction solvent of PE:95% alkali ethanol containing
2% NaOH (4:6). More than 96% nicotine and solanesol in the mate-
rial were extracted with 1.0 MV or 2-fold excess solvent of the
material (v/w) through a cyclic CCE procedure in both small-scale
and enlarged-scale experiments. The extraction solution was
automatically separated into an ethanol-aqueous phase containing
98% nicotine and an ether phase containing 96% solanesol at pH
2.0. The ethanol-aqueous phase was vacuum-concentrated to
recover the ethanol, and 99% purity nicotine was obtained by PE
fractionation at pH 10.0. Solanesol in the ether phase was purified
to 93.1% by a single step silica gel column chromatography. The
procedure used simple equipment, and minimum amount of sol-
vents. All the processes were completed at room temperature,
and all solvents used in the extraction, separation and purification
processes were completely recovered for reuse without environ-
mental pollution.
Acknowledgements
This work is supported by the Special Research Fund for
University Ph.D. Programs (Program 20134407120004), the Open
Programs of the Provincial Key Laboratory of Green Processing
Technology and Natural Products Safety (Program 201307) and
the Special Research Fund of SCNU for Young Teachers (Program
13KJ10).
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