Continuous counter current extraction, isolation and determination of solanesol in Nicotiana tobacum L. by non-aqueous reversed phase high performance liquid chromatography

doi:10.1016/j.jpba.2007.10.014

Journal of Pharmaceutical and Biomedical Analysis

Volume 46, Issue 2, 22 January 2008, Pages 310-315

https://doi.org/10.1016/j.jpba.2007.10.014 Get rights and content

Abstract

A method of continuous counter current extraction in a large-scale production of solanesol from tobacco leaves was developed. The crude extract containing 15–20% solanesol was subjected to a series of steps, viz., saponification, solvent recrystallization and column chromatography. The pure material was characterized by FT-IR, ESI-MS, ¹H NMR and ¹³C NMR spectrometry. The analysis was carried out by a simple and rapid non-aqueous reversed-phase high performance liquid chromatographic method on a Hypersil BDS C₁₈ column (250 mm × 4.6 mm, particle size 5 μm) with isopropyl alcohol–methanol (60:40, v/v) as mobile phase and detection at 215 nm. The product purity was between 95 and 98% (w/w) as determined by HPLC.

Introduction

Solanesol a naturally occurring trisesquiterpenoid (C₄₅) alcohol of tobacco is one of the important precursors of the tumorigenic polynuclear aromatic hydrocarbons (PAHs) of tobacco smoke. Reduction of its levels in tobacco, leads to safe smoking products due to reduced PAH levels in cigarette smoke. It is also the starting material for many high-value biochemicals, including coenzyme Q10 and Vitamin-K analogues [1] as a starting material for Q10, it is used in treatment of different cancers. Coenzyme Q10 is well known not only to reduce the number and size of tumors but also improve cardiovascular health [2], [3]. Solanesol itself could be used as an antibiotic, cardiac stimulant and lipid antioxidant. At present clinical trails are under progress to explore its use as an anticancer drug. There is a great demand for solanesol for production of Q10 and other uses. Thus, its isolation not only reduces the risks of PAH from tobacco smoke but also makes use of it as a starting material in synthesis of several value added products such as Q10 and other analogues. Therefore, isolation of solanesol from tobacco is gaining a lot of importance in recent years.

Solanesol is present in the lamina of tobacco leaves while absent from the stem and stalk [4]. The content of solanesol in tobacco depends upon a number of factors. It varies from 0.3 to 3.0% according to the type and variety of tobacco, duration of growth and method of curing [5], [6]. A substantial portion of solanesol exists in the form of fatty acid esters due to which proper curing and saponification play an important role in converting them in to free solanesol [7]. Tobacco also contains several other organic compounds that can be easily co-extracted with solanesol and interfere with subsequent separation and purification processes [8]. One of the key problems in extraction of solanesol from tobacco is the selection of a suitable solvent for maximum yield and purity. As the solanesol lies in the cellular chloroplast of the tobacco leaves, not only the solubility but also penetrability of the solvent is very important for complete extraction. Further its separation from the crude extract and purification pose several problems because of the presence of closely related fatty acids, alcohols, alkaloids, tobacco pigments, tar and other organic impurities. The food and pharmaceutical grade solanesol has to be of highest purity for quality, safety and efficacy of the finished products. Therefore, it is quite important to develop processes that can selectively separate and purify solanesol from the crude extracts of tobacco leaves. Several methods were described in the literature for extraction, isolation and purification of solanesol from tobacco [9], [10], [11], [12], [13]. Most of the methods involve multiple step procedures, which are non-specific, quite tedious and time consuming. Generally, maceration, percolation, ultra sonication, soxhlet and bubble column were used for extraction of phytochemicals from the plant materials [14], [15]. The first two techniques are not only time consuming but also give low yields of the desired products. Microwave-assisted extraction (MAE) coupled with saponification was reported to be effective for extraction of solanesol from tobacco leaves [16]. However, MAE and ultrasound sonication are useful for high value added products but consume high energy for commercial production. Soxhlet has limited analytical applications and not suitable for handling of bulk quantities of tobacco. Tang et al proposed an extraction procedure with petroleum ether under reflux at 50 °C followed by silica gel column chromatography for isolation and purification of solanesol from tobacco leaves [17]. However, the heat-reflux processes involve lengthy operations, bulk amount of solvents and ultimately thermal decomposition of the target compounds. Recently high-speed countercurrent chromatography (HSCCC) for isolation of solanesol from the crude extracts of tobacco was reported [18]. Here, the crude extract instead of raw tobacco was used as a feedstock for purification of solanesol. The purity of solanesol thus obtained was lees than 95%. A slow rotary counter current chromatography (SRCCC) involving a non-aqueous two-phase solvent system of sunflower oil-ethanol was also used to produce food grade solanesol in a commercial scale [19]. However, the process is not cost effective and the purity of solanesol was only 26%.

Reliable methods for determination of solanesol in tobacco are important for classification of different grades of tobacco according to their quality. A number of gas chromatographic methods for determination of solanesol in tobacco were reported [20], [21], [22], [23], [24]. The low volatility and poor FID response of solanesol render the technique unsuitable [20]. The sample preparation also involves a number of time-consuming derivatization steps [4], [21], [22]. Sheen et al. reported a packed column GC method involving lengthy extraction procedures and hydrogenation of solanesol [23]. The other methods include gravimetry [24] coulometry [25] and thin layer chromatography (TLC) [24]. HPLC with various detectors including UV, RID, ELSD and MS was used to determine solanesol. Most of the methods reported before 2006 were in normal phase mode with UV detection [26], [27], [28]. Until the early 2007, not even a single reversed-phase HPLC method for determination of solanesol in tobacco was reported. Recently, Zhoua et al. reported a RP-HPLC method using ELSD as a detector for determination of solanesol in tobacco [29]. ELSD is not only a specialized detector but also requires a large volume of nebulizer gas of high purity. It makes the method unsuitable for routine analysis solanesol because of the cost ineffectiveness. However, reversed phase HPLC with UV detection is often preferred not only because of its higher sensitivity but also wide availability and suitability. Quite recently, Chen et al. reported RP-HPLC with UV and ESI-TOF/MS determination of solanesol in the crude extracts of tobacco leaves [30]. The major drawback of this method lies in detection. The analytes were monitored at 202 nm by PDA where the acetonitrile used as one of the mobile phase solvents generally interferes. The use of such a short wavelength UV also produces baseline artifacts. Due to the increasing demand for solanesol, a convenient and rapid method for determination as well as isolation of solanesol from tobacco is highly needed.

In the present investigation: (i) a simple protocol involving the use of a continuous counter current extraction followed by saponification, solvent crystallization or column chromatography for isolation of solanesol from Nicotiana tobacum L. was developed. It was compared with soxhlet extraction in terms of efficiency and recovery. Further, a simple non-aqueous RP-HPLC with UV method for determination of solanesol in Nicotiana tobacum L. was described.

Section snippets

Materials and reagents

All the reagents were of analytical-grade unless stated otherwise. HPLC-grade isopropyl alcohol and methanol (Ranbaxy, SASNagar, India) were used. Dried tobacco received from local industries in Hyderabad, India and farmers of Nigeria, West Africa were used.

Counter current extraction

The counter current continuous extraction was carried out in nine stages. Initially the dried leaves were powdered in pulveriser and particles of 2–3 mm size were pelletized of size 12 mm × 24 mm using steam. The pellets were then passed to the

Extraction of solanesol

It is always economical to produce solanesol first in the form of a crude extract in a continuous counter current extractor. The extract was further purified to high-grade solanesol by different methods. In the counter current extraction process, steam was introduced in to the pellitiser so that, the powder absorbs around 5–8% moisture while forming the pellets. The moisture helps in the separation of water-soluble compounds and increases the content of solanesol in the extract. The water was

Conclusions

An economical and efficient protocol for isolation of solanesol from tobacco using counter current extraction, followed by column chromatography, saponification and recrystallization was described. High purity of 95–98% solanesol was produced using common laboratory chemicals. The continuous counter current extraction is more suitable for isolation of solanesol on a large scale. In addition, a simple and rapid method for separation and determination of solanesol from tobacco using non-aqueous

Acknowledgement

The authors thank Dr. J.S. Yadav, Director Indian Institute of Chemical Technology for encouragement and permission to communicate the results for publication.

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Increasing the processing time further did not increase the yield and in fact, decrease the yield to 0.65 wt% (p < 0.001). Similar results were observed in previous studies in which the amount of solanesol extracted was decreased by extending maceration period more than 4 hours [4, 5, 37]. This effect might be due to degradation or complexation of solalnesol in the solution.
Solanesol, the precursor for the synthesis of coenzyme Q10, is currently recovered from tobacco leaves by conventional extraction techniques that require multiple purification steps and a large amount of organic solvents. We recently identified tomato leaves as an alternative source of solanesol and hypothesized that a high-pressure CO₂ extraction could be used as a clean extraction process. The effect of CO₂ pressure and temperature on the extraction of solanesol was determined to achieve high yield and purity. It was found that solanesol could be extracted efficiently by subcritical CO₂ at 25 °C from tomato leaves. The extract contained 40% solanesol and other active compounds such as vitamin K1. A higher level of purity of 93% was achieved using a secondary purification step. Different conventional methods for solanesol extraction was compared to determine the most efficient technique for production of solanesol from tomato leaf. The highest yield of solanesol was achieved at nearly 1% dry weight with using subcritical CO₂, which was superior to conventional methods.
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The extraction solution was automatically separated into PE and ethanol-aqueous phases, which not only completely separated solanesol and nicotine but also made the solvents be easily recovered 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 chromatography, 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.
The tobacco (Nicotiana tabacum L.) cultivation and cigarettes manufacture industries discard huge amount of waste tobacco materials that have strong nicotine smell and contaminate the environment. A high-efficient procedure was developed for simultaneously extracting nicotine and solanesol from the waste tobacco leaf vein through the column-chromatographic extraction (CCE), followed by 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). Greater than 96% extraction efficiency for both nicotine and solanesol was obtained with a 2-fold excess solvent of the material (v/w) through a cyclic CCE procedure in small-scale and scaled-up experiments. The extraction solution was 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 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 processes 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.
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The production of biopharmaceutical proteins using plant-based systems has recently become economically competitive with conventional expression platforms based on microbes and mammalian cells, but downstream processing remains a significant cost factor. Here we report that, depending on the protein expression level, production costs for biopharmaceuticals made in plants can be reduced by up to 30% if a juice extractor is used instead of a blade-based homogenizer or blender. Although the extraction efficiency is lower, combining extraction and solid–liquid separation into a single operation reduces the extract volume by 80%, which achieves savings of ∼60% for downstream consumables and labor. Additionally, juice extraction can easily be scaled-up to process several tons of biomass per day and its continuous mode of operation simplifies downstream processing steps because the volume of storage tanks and the duration of hold times are reduced. The juicer setup is also compatible with flocculation and to some extent with leaf blanching, which increase the efficiency of extract clarification and product purification, respectively. The use of juicers can therefore significantly increase the competitiveness of plant-based production platforms.
Expanded separation technique for chlorophyll metabolites in Oriental tobacco leaf using non aqueous reversed phase chromatography
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The chlorophyll metabolites bound to solanesol in plants have not been reported yet. Meanwhile, tobacco leaves are known for including a large amount of solanesol [29–33] which is increasingly accumulated just before harvesting or during the curing process [14]. Therefore, they were considered tobacco specific components.
An improved separation method for chlorophyll metabolites in Oriental tobacco leaf was developed. While Oriental leaf still gives the green color even after the curing process, little attention has been paid to the detailed composition of the remaining green pigments. This study aimed to identify the green pigments using non aqueous reversed phase chromatography (NARPC). To this end, liquid chromatograph (LC) equipped with a photo diode array detector (DAD) and an atmospheric pressure chemical ionization/mass spectrometer (APCI/MSD) was selected, because it is useful for detecting low polar non-volatile compounds giving green color such as pheophytin a. Identification was based on the wavelength spectrum, mass spectrum and retention time, comparing the analytes in Oriental leaf with the commercially available and synthesized components. Consequently, several chlorophyll metabolites such as hydroxypheophytin a, solanesyl pheophorbide a and solanesyl hydroxypheophorbide a were newly identified, in addition to typical green pigments such as chlorophyll a and pheophytin a. Chlorophyll metabolites bound to solanesol were considered the tobacco specific components. NARPC expanded the number of detectable low polar chlorophyll metabolites in Oriental tobacco leaf.
Solanesol: Added value from Solanaceous waste
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Purification of solanesol has focussed on using tobacco leaves as the starting material. Many protocols have been published, including conventional heat reflux extraction (Keca et al., 1997), solid phase extraction (Tang et al., 2007), ultrasound- (Keca et al., 1997; Chen et al., 2007), microwave-assisted extraction (Zhou and Liu, 2006), counter-current chromatography (Zhao and Du, 2007; Rao et al., 2008) and supercritical fluid extraction (Ruiz-Rodriguez et al., 2008). The curing process has been shown to enhance the release of bound solanesol in the form of esters from flue-cured tobacco products (Severson et al., 1977; Chamberlain et al., 1988; Liu et al., 1998) and wastes (Zhao et al., 2005; Qiu et al., 2007).
Isoprenoids, also known as terpenoids, are the largest and oldest class of natural products known. They are comprised of more than 40,000 different molecules all biosynthetically related via a common five carbon building block (isopentenyl). Many isoprenoids are of commercial interest and are used as fragrances in cosmetics and flavours, colorants and nutritional supplements in foods and feeds as well as being phytomedicines. Their industrial relevance also means they are compounds of high value with global markets in the range of $1 billion per annum. Solanesol is a 45-carbon, unsaturated, all-trans-nonaprenol isoprenoid of high value. Recently this molecule has received particular attention because of its utility, both in its own right and as a precursor in the production of numerous compounds used in the treatment of disease states. Instability in supply and spiralling costs have also lead to the search for sources. In this article existing sources and the potential strategies and tools available to create sustainable biosources are reviewed.

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Continuous counter current extraction, isolation and determination of solanesol in Nicotiana tobacum L. by non-aqueous reversed phase high performance liquid chromatography☆

Abstract

Introduction

Section snippets

Materials and reagents

Counter current extraction

Extraction of solanesol

Conclusions

Acknowledgement

Clin. Biochem.

J. Chromatogr.

J. Chromatogr. A

Biochem. Eng. J.

J. Chromatogr. A

Sep. Purif. Technol.

J. Chromatogr. A

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Pharm. Biomed. Anal.

J. Chromatogr. A

J. Pharm. Biomed. Anal.

J. Label Compd. Radiopharm.

Cardiovasc. Res.

J. Yunnan Univ.