Stewart Postharvest Review

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Aug 1, 2007 - lytical sector are not reflected in these standard works. ... Threshing/picking/crushing: removal of seeds, flowers, ... ple is comminuted with a homogeniser directly in the solvent ... liquid-liquid extraction or a solid phase extraction (SPE) is not possible. However, there are exceptions when the analyte is.
Stewart Postharvest Review An international journal for reviews in postharvest biology and technology

Analytical techniques for medicinal and aromatic plants Hans Krüger and Hartwig Schulz* Federal Centre for Breeding Research on Cultivated Plants, Institute of Plant Analysis, Erwin-Baur-Str.27, D-06484 Quedlinburg, Germany

Abstract The analytical techniques that are applied for medicinal and aromatic plants are used particularly for the analysis of secondary substances, minor components in plants that are distributed very inhomogenously. Therefore, the main analytical problems that exist are related to correct sample preparation and separation of the analytes. This article reviews different methods of operation such as wet milling, extraction and distillation, and describes some special micro and headspace techniques. This paper also covers modern chromatographic techniques and special possibilities within the range of the near-infrared, attenuated total reflection mid-infrared and Raman spectroscopy that are used for non-destructive and rapid analysis of medicinal and aromatic plants. Keywords: sample preparation; distillation; extraction; microtechniques; headspace; vibrational spectroscopy

Abbreviations Accelerated Solvent Extraction ASE Attenuated Total Reflection ATR Fourier Transform FT Gas Chromatography GC High Performance Liquid Chromatography HPLC HPTLC High Performance Thin Layer Chromatography Infrared IR Mass Spectrometry MS Near Infrared NIR Stir Bar Sorptive Extraction SBSE Simultaneous Distillation-Extraction SDE Supercritical Fluid Extraction SFE Solid Phase Dynamic Extraction SPDE Solid Phase Extraction SPE Solid Phase Microextraction SPME

Introduction

*Correspondence to: Hartwig Schulz, Federal Centre for Breeding Research on Cultivated Plants, Institute of Plant Analysis, Erwin-Baur-Str.27, D-06484 Quedlinburg, Germany

The nature of plants and substances, their use and activity are described in pharmaceutical biology and chemistry of natural products texts [1–4]. Special handbooks [5–7], technical standards (eg, ISO 6571 or DIN 10 228) and pharmacopoeias provide reviews of standardised analytical methods, but often rapid technological developments in the analytical sector are not reflected in these standard works. Therefore, starting from standard methods, this article de-

Stewart Postharvest Review 2007, 4:4 Published online 01 August 2007 doi: 10.2212/spr.2007.4.4 © 2007 Stewart Postharvest Solutions (UK) Ltd.

The active compounds in medicinal and aromatic plants are mainly secondary substances that occur as complex mixtures. They are often used in medicinal and cosmetic products, as well as in food supplements. The analytical techniques applied for the characterisation of medicinal and aromatic plants should be suitable for separation, structure elucidation and quantification of the related substances. The main components are not necessarily the active substances, as quite often minor constituents are more responsible for physiological effects and determine the quality of these plants. The chemical nature of secondary substances can be very different, extending from monoterpenes to plant resins. In addition, the following classes of natural substances are of importance: sesquiterpenes, diterpenes and polyterpenes, phenylpropanes, amaroides, glycosides, saponins, alkaloids, glucosinolates, anthracenes, flavonoids, coumarins, tannins, lipoids, lectins, and others.

Krüger and Schulz / Stewart Postharvest Review 2007, 4:4

Figure 1. Tube and vibrating mill for comminution of individual fennel fruits and extraction of essential oil components.

− − −

fennel seed

Fennel seed

scribes specific developments in the separation and quantification of secondary compounds. Techniques for structure elucidation are deliberately not considered. In addition, hyphenated techniques, which are important for detection of substances in highly complex natural mixtures, are described only if they are important for the characterisation of individual plant species. In order to answer the question of which technique is the most suitable, several criteria, besides the plant matrix, must be considered. In this context the following aspects must be noted: − The nature of the sample: small/big, dry/fresh, liquid/ solid, homogeneous/inhomogeneous − The nature of the analyte: volatile/non-volatile, stable/ unstable, polar/non-polar − The specific problem under consideration: quantification in a homogeneous sample, distribution within a plant, in a part of a plant or in a plant cell. − Is it necessary to perform non-destructive measurements (eg, in the case of seed samples in the breeding process)? All aspects are fundamental for sample preparation, the separation of analytes, and their identification and quantification.

Sample preparation Most medicinal and aromatic plants produce a wide spectrum of secondary substances. After harvest, the first step in sample preparation is usually the drying process. For essential oil plants particularly, the danger exists that a large portion of the active substances will be modified in the postharvest process [8–10], and also that the concentration of certain non-volatile substances may be influenced. For example, the reduction of morphine in Papaver during the preparation process is well known, and is a result of the alkaloid reacting with plant phenols and organic acids. Besides the drying process, further sample preparation steps have to be performed in the laboratory and in subsequent industrial processing, and additional loss of active substances may be observed. These include: − Threshing/picking/crushing: removal of seeds, flowers, leaves, etc from stalks

The processing procedure used is chosen according to the plant organ in which the active ingredient is accumulated. In literature, mechanical and drying processing of medicinal plants are mostly treated peripherally and explicitly in only a few cases [11–13]. Comminution losses can often be reduced by cryo-milling [14]. A further approach to avoid preparation losses exists in wet milling of drugs [15], in which the sample is comminuted with a homogeniser directly in the solvent and the extraction of components takes place at the same time. This step can be also miniaturised; for this 2 mL tubes are clamped in a vibrating mill – the tube contains a small quantity of material (3–10 mg), approximately 1 mL solvent and a grinding ball (Figure 1). The drug is comminuted by the vibration process and the active substances are dissolved simultaneously (Figure 1). In this way the essential oil components of individual fennel fruits were determined and the distribution of the volatile fraction was calculated for a single fennel umbel (Figure 2).

Separation of the analyte If a homogeneous sample can be obtained, isolation of the secondary substances can be performed by extraction. Because vegetable samples are generally solids, direct application of liquid-liquid extraction or a solid phase extraction (SPE) is not possible. However, there are exceptions when the analyte is present in an aqueous phase, eg, in fruit juices [16] or in aqueous distillation fractions [17]. Sometimes a hydrolytic decomposition can precede the extraction. This applies to those cases with unstable analytes, which are subjected to chemical decomposition during processing. Examples of this are the enzy-

Figure 2. Essential oil content of 99 fennel fruits of one fennel umbel (cultivar ‘Berfena’) 30 24

25

Number of seeds

Solvent solvent grinding Grindingball ball

Separation: separating from stalks and other residues Size reduction: producing smaller particles (eg, by cutting or milling) Classification: sorting the particles into different grades

24 21

20 15

15 10

7

7

5 1

0 4-5%

5-6%

6-7%

7-8%

8-9%

9-10%

10-11%

Oil content groups 2

Krüger and Schulz / Stewart Postharvest Review 2007, 4:4

Figure 3. Distillation-extraction in an SPE-cartridge.

Solid phase (RP-18)

Drug Filter

can be improved by adding polar modifiers such as methanol, acetone, hexane or dichloromethane. Accelerated solvent extraction (ASE) has been in use in the analytical sector for some years now. ASE uses liquidextracting agents such as pentane, ethyl acetate, acetone and others for extraction of solid samples. Extraction takes place at temperatures from room temperature up to 200°C and is usually performed at pressures of 0.3–20 MPa in order to handle the solvent above its boiling point as liquid [23].

Spring Water

matic hydrolysis of glucosinolates in Brassicaceae [18] and of cysteine sulphoxides in Allium species [19]. Extracting plant material with organic solvents is the most common extraction technique. Soxhlet extraction is chiefly applied to the separation of relatively non-volatile analytes from solid samples [7]. The advantage of this method is high yield, provided by continuous extraction of the sample with fresh extractant. Disadvantages include the considerable amount of time required (typically several hours to several days), the thermal stress to which analytes are subjected and the fact that the analytes are obtained in very dilute form in a solvent and must therefore be concentrated in a subsequent step. Often any fat present in the sample is co-extracted, and must therefore be removed separately. As already mentioned, the combination of wet milling and extraction offers some advantages. In this context it is aimed at ensuring rapid transition of the analyte into the solvent. Another approach is to apply supercritical fluid extraction (SFE). This method is also used in industry for isolation of various aromatic products [20, 21]. The isolation of carnosolic acid, a natural antioxidant from Rosmarinus officinalis, is a typical example of this gentle extraction method. Supercritical CO2 is the most frequently used extractant for SFE. It has the advantage of being chemically rather inert, and its critical temperature is low, so it is valuable for the extraction of such labile analytes as steroids, fragrances and antioxidants [22]. The low critical temperature and pressure (31.1°C and 7.37 MPa) opens the way to a relatively broad range within which the solvency can be varied. Other advantages of CO2 as an extractant include low toxicity, high purity and low cost. The principal disadvantage of CO2 is its relatively low polarity. Solvent power, with respect to polar analytes,

At increased temperatures extraction kinetics are strongly accelerated; the desorption of the analytes from the matrix and the dissolution process run substantially faster than at room temperature. In addition, the increased temperature permits better solubility of the analyte in the extracting agent and also has a substantial influence on extraction yields. In this process, which is suitable for automation, pressureresistant high-grade steel extraction cells with volumes of 1–100 mL are used. As in the case of soxhlet extraction, the ideal sample is dry, homogeneous and equally milled. Herbal samples often possess increased water contents. In these cases polar water-miscible solvents (eg, acetone or methanol) or mixtures of different polar solvents (eg, dichloromethane/ acetone or hexane/acetone) are used. In addition, desiccating agents have been selected, and for extracting vegetable samples in particular diatomaceous earth has been applied [24]. There are numerous applications for medicinal and aromatic plants, eg, for vanilla [25], chili varieties [26] or St. John’s wort [27]. For extraction with polar solvents or of damp samples, microwave assisted extraction also comes into consideration [28], eg, for the extraction of volatiles from fresh aromatic herbs. An alternative approach to separate volatile compounds from liquid and solid samples is distillation. Steam distillation is an effective way to separate volatile substances, eg, phenols [29] and essential oils from the plant matrix. The standard method for isolation of essential oils, described in the European Pharmacopeia of 2005 [30], is hydrodistillation. Individual monographs have been published for the following essential oils and essential oil drugs: Achillea millefolium, Angelica archangelika, Anthemis nobilis, Artemisia absinthium, Capsicum annuum, Carum carvi, Cinnamomum cassia (oil only), Cinnamomum verum (oil only), Cinnamomum zeylanicum (drug and oil), Citrus aurantium (drug and oil), Citrus limon (oil only), Citrus sinensis (oil only), Coriandrum sativum (drug and oil), Curcuma xanthorrhiza, Cymbopogon winterianus (oil only), Eucalyptus globulus (drug and oil), Filipendula ulmaria, Foeniculum vulgare (drug and oil), Illicium verum (drug and oil), Juniperus communis (drug and oil), Lavandula angustifolia (drug and oil), Levisticum officinale, Matricaria recutita (drug and oil), Melaleuca alternifolia (oil only), Melissa officinalis, Mentha arvensis (oil only), Mentha piperita (drug and oil), Myristica fragrans (oil only), Pimpinella anisum (drug and oil), Pinus pinaster (oil only), Rosmarinus officinalis (drug and oil), Salvia offici3

Krüger and Schulz / Stewart Postharvest Review 2007, 4:4

Figure 4. Correlation between essential oil values of fennel fruits determined by hydrodistillation and solvent extraction. 25,00 25

20,00 20

y == 0.9583x 0,9583x–- 0.8165 0,8165 2

Distillation Distillation

= 0,9367 R2 = R 0.9367 15,00 15

10,00 10

5,00 5

0,00 0 0,00 0

5,00 5

10,00

10

15,00 15

20,00 20

25,00 25

Extraction Extraction

Figure 5. The relative deviation of essential oil values from the mean as a function of the fennel fruit number.

10 8 6

Relative deviation deviation (%) relative (%) 5.6 4.5

2.6

4

y = 49.506x -0.8571 R2 = 0.889

2.9 1.5

1.9

50

60

70

-1.8

-1.4

1.3

2

0.8

1.0

80 -1.0

90 -0.8

0 -2

10

20

30

-1.9

-4 -3.6 -6 -8

40

-3.9

-3.1 num ber of of seeds seeds Number

y = 2.6953Ln(x) - 12.76 R2 = 0.9396

-7.1

-10

4

Krüger and Schulz / Stewart Postharvest Review 2007, 4:4

nalis, Salvia sclarea (oil only), Salvia triloba, Syzygium aromaticum (drug and oil), Thymus serpyllum, Thymus vulgaris (drug and oil), Valeriana officinalis and Zingiber officinale. Steam distillation offers the special advantage that the analyte is recovered in a nearly matrix-free condition, although it is sometimes diluted with a large amount of water from which it must subsequently be separated and concentrated. SPE is particularly useful for this purpose [17]. Another classical method for separating steam volatile substances during a distillation process is the Likens–Nickerson extraction [31] or simultaneous distillation-extraction (SDE). This apparatus also allows circulatory distillation and permits, as a major advantage, a many thousand-fold concentration of volatiles from aqueous media in one step. The equipment has found wide application in numerous laboratories and several modified versions have been constructed [32]. For execution of this distillation method, special flasks with a volume of at least 250 mL are used. Thus, the minimum weighed portion normally consists of approximately 5 g of plant material. For characterisation of single plants or parts of plants this amount is sometimes too large. Therefore, attempts to provide miniaturisation of hydrodistillation and SDE [33, 34] have been proposed, but they have limitations. Microdistillation using capillaries [35, 36] and distillation-extraction using solid phase cartridges (Figure 3) [37] are the only new approaches for making a distillative separation possible also from small plant samples. For the investigation of seed samples obtained from plant breeding experiments, wet milling methods in solvents were applied and the essential oil value was calculated as the sum of all detected single volatile components. For this purpose, gene bank material, which exhibited a large variability, was examined previously and it was investigated whether these sum values correlate with those obtained by using the classical distillation method. It was shown that this procedure can be successfully applied for fennel fruit samples (Figure 4). These extraction values served as the basis for nondestructive, spectroscopic methods, which are described at the end of this paper. Extractions were accomplished with 200 mg of seed material. This quantity was necessary to limit the standard deviation, which results particularly from the inhomogeneity of the sample, to less than 3% (Figure 5). For characterisation of single plants the amount cannot be smaller. The basis for investigating deviation from the average was a data record of 99 single fruit analyses. Fruits were prepared by applying the micromethod described above (Figure 1). Micromethods are also presented as headspace and purge and trap methods. Modern microtechniques use often solid phases for accumulation of analytes (solid phase microextraction SPME, solid phase dynamic extraction - SPDE, stir bar sorptive extraction - SBSE). Although direct connection of the analytes onto the solid phase of the fibre is also possible in the liquid phase [38], the accumulation of volatile substances in many cases takes place in the gas phase by a static head-

space sampling, because this method enables easy separation from the matrix, as well as from other non-volatile secondary substances. SPME in combination with gas chromatographymass spectrometry (GC-MS) is used globally as a monitoring technique for volatile compounds in medicinal and aromatic plants [39, 40]. The principle of accumulation is similar for SPME, SPDE and SPSE. In all cases the analyte is fixed by a special adsorbent material (eg, PDMS; fibre – SPME, needle – SPDE, stir bar – SBSE). SPDE and SBSE possess a higher amount of polymer and thus a higher capacity and a lower detection limit. Examples of application in the area of medicinal and aromatic plants are described by Bicchi et al. [41]. Purge and trap methods (dynamic headspace sampling) are described as closed-loop stripping or as “pull” or “pushpull” systems. In these sampling methods, a continuous air stream flows through the sample vial as a carrier gas. While the analytes are trapped on adsorbents, the carrier gas is circulated (closed loop) or purged out of the sample vial (pull). A large number of different adsorbent materials are available and several reviews describe the applications [42–44].

Quantification of active substances All distillation, extraction and accumulation techniques are basis for good separation, identification and quantification by chromatographic methods. Many publications describe classical chromatographic techniques (high performance thin layer chromatography [HPTLC], GC and high performance liquid chromatography [HPLC]) in the field of medicinal and aromatic plants. HPTLC has been established as an analytical method for medicinal and aromatic plants for a long time. Most pharmacopoeias contain directions for examining the identity of medicinal and aromatic plants, but often these methods are not up-to-date. Modern methods for qualitative and quantitative HPTLC use automatic spray-on techniques for sample application, horizontal development chambers, computer controlled densitometric chromatogram evaluation, and electronic pattern documentation [45–47]. Recently, the analysis of medicinal plants by thin layer chromatography was reviewed by Gocan et al. [48]. Capillary GC is the standard technique for detection of volatiles [49, 50]. Samples are either directly injected as solvent extracts into the heated injector in split or splitless mode or desorbed from the adsorbent by placing it in a thermal desorbtion tube, heated to 250–300°C. Volatiles are commonly separated on fused silica capillary columns with different stationary phases such as the non-polar dimethyl polysiloxanes (eg, DB-1, DB-5, CPSil 5), and the more polar polyeth ylene glycol polymers (eg, Carbowax 20M, DB-Wax). Volatile substances can be analysed by different detectors. Flame ionisation detectors are most commonly used because of their wide linear dynamic range, their very stable response, high sensitivity and favourable price. MS detectors provide information on the retention time of each compound and the individual mass spectrum. Quantification is possible in full scan or selected ion monitoring mode, but requires calibration for each individual compound. For identification of compounds 5

Krüger and Schulz / Stewart Postharvest Review 2007, 4:4

Figure 6. Near infrared spectroscopy correlation for the essential oil content (mL/100g) in air dried fennel fruits.

16,00 16

14,00 14

1,0038x+ +0.7207 0,7207 yy==1.0038x 2 R2 = 0.9514 R = 0,9514

NIRS prediction NIRS-prediction

12,00 12

10,00 10

8,00 8

6,00 6

4,00 4

2 2,00

0,00 0 0,00 0

2,00 2

4,00 4

6,00 6

8,00 8

10,00 10

12,00 12

14,00 12

Extraction Extraction

Abbreviation: NIRS, near infrared spectroscopy

in GC–MS analysis, predictions can be obtained from mass spectral libraries such as Wiley and the National Institute of Standards and Technology. In addition to the identification of volatile compounds, it is often important to determine their chirality. Modern chiral columns possess a high separation efficiency not only for enantiomers, but also for non-chiral substances. More difficult separation problems may be solved sometimes by two-dimensional techniques (non-chiral – chiral) or tandem MS (MS-MS). Concrete directions for analysis of chiral volatiles have been described by König [51], Bicchi [52], Mosandl [53], and others. HPLC is the standard technique for separation of non-volatile substances. The research in this field focuses on development of validated methods by single or hyphenated HPLC. In most cases reversed-phase HPLC methods were established for separate or simultaneous determination of sesquiterpenes, diterpenes and polyterpenes, phenylpropanes, amaroides, glycosides, saponins, alkaloids, glucosinolates, anthracenes, flavonoids, coumarins, tannins, lipoids, lectins, and others. There exists a wide literature pool for method development including experimental conditions and sample preparation. A review of the analysis of medicinal plants by HPLC was published by Cimpan et al. in 2002 [54].

Applications of vibrational spectroscopy in the analysis of medicinal and aromatic plants Sample preparation, which provides a representative analytical sample, and separation of the analyte, which enables a trouble-free analysis with high accuracy and low standard deviation, are prerequisites for successful application of rapid spectroscopic determinations in the analysis of medicinal and aromatic plants. Vibrational spectroscopic methods offer the possibility to rapidly analyse herbal samples without destruction. Advantages exist, especially for seed samples, because after the analysis all seeds are available for further breeding processes. Short reviews of near-infrared (NIR), infrared (IR) and Raman spectroscopic methods applied to the analysis of various medicinal plants are available [55–57]. NIR spectroscopy as a routine technique was established in the early 1980s when efficient chemometric algorithms were successfully introduced. Whereas NIR spectroscopy data can be interpreted only by application of chemometric algorithms, IR and Raman spectra obtained from individual plant samples are mostly well-structured and present characteristic key bands (Table 1). 6

Krüger and Schulz / Stewart Postharvest Review 2007, 4:4

Table 1. Assignment for the most characteristic Raman and IR bands of some essential oil components.

Compounds Carvacrol 1,8-Cineole

FT-Raman (cm-1)

Assignment

ATR-IR (cm-1)

Assignment

1,623 760

ν (C=C) δ (Ring)

811

652

δ (Ring)

1,374 1,214 1,079 984 843

δsym (CH3(CO)) νas (C-O-C) νs (C-O-C) ω (CH2)

ω (C-H)

Citronellal

1,725 1,674 1,382

ν (C=O) ν (C=C) δsym (CH3)

1,725 1,116

ν (C=O)

Citronellol

1,674 1,382

ν (C=C) δsym (CH3)

1,377

δ (C-O-H)

p-Cymene

1,613 1,208 804

ν (Ring) δ (Ring) δ (Ring)

1,515 813

ω (C-H)

Geranyl acetate

1,679

ν (C=C)

1,738 1,365 1,227 1,021

ν (C=O) δsym (CH3(C=O)) νas (C-O-C) νs (C-O-C)

Limonene

1,678 1,645 760

ν (Cyclohexene C=C) ν (Ethylene C=C) δ (Ring)

1,644 886

ν (Ethylene C=C) ω (C-H)

Myrcene

1,672 1,634 1,293

ν (C=C) ν (C=C) τ(CH2)2

1,637 1,595 989 890

ν (C=C) ω (CH2) ω (C-H)

Phenylethyl phenylacetate

1,605 1,203 1,003

ν (Ring) δ (Ring) δ (Ring)

1,736 1,131 816 698

ν (C=O) ν (C-O-C) ω (C-H) δ (Ring)

α-Pinene

1,659 666

ν (C=C) δ (Ring)

1,658 886 787

ν (C=C) ω (C-H)

β-Pinene

1,643 645

ν (C=C) δ (Ring)

1,640 873

ν (C=C)

Sabinene

1,655 652

ν (C=C) δ (Ring)

1,653 861

ν (C=C)

cis-Sabinene hydrate

748

δ (Ring)

926

ω (CH2)

trans-Sabinene hydrate

755

δ (Ring)

920

ω (CH2)

Thymol

740

δ (Ring)

804

ω (C-H)

Terpinen-4-ol

1,679 730

ν (Cyclohexene C=C) δ (Ring)

924 887

ω (CH2) ω (C-H)

α-Terpinene

1,611

ν (Conjugated C=C)

823

ω (C-H)

γ-Terpinene

1,701 756

ν (Nonconjugated C=C) δ (Ring)

947 781

ω (CH2) ω (C-H)

Abbreviations: ATR-IR, attenuated total reflection infrared spectroscopy; FT, Fourier transform 7

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Table 2. Range, mean and NIRS correlation statistics for the essential oil content (mL/100g) and composition (%) in the dried leaves of Mentha piperita (n=82). Range

Mean

SECV

R2

Essential oil content

0.63–3.63

2.13

0.19

0.93

Limonene

0.00–6.47

1.25

0.62

0.82

1,8-Cineole

1.15–6.04

3.59

0.47

0.80

Menthofuran

0.00–4.20

1.01

0.64

0.62

10.10–56.90

30.91

4.00

0.88

2.02–8.44

4.16

0.98

0.53

15.90–58.60

38.13

3.93

0.86

Menthyl acetate

0.77–7.89

3.78

1.11

0.57

Pulegone

0.00–5.77

0.85

0.63

0.64

Piperitone

0.41–2.38

1.25

0.34

0.60

Parameter

Menthone Isomenthone Menthol

Abbreviations: NIRS, near infrared spectroscopy; R2; multivariate coefficient of determination; SECV, standard error of cross validation.

Based on such marker bands produced by individual secondary substances, spectroscopic analyses in principle allow the discrimination of different species and even chemotypes among the same species. In many cases quantitative determination of the main components is possible by applying the partial least square algorithm, and the ability to rapidly monitor various plants makes it possible to efficiently select high-quality single plants from wild populations, as well as from progenies of crossing experiments. Furthermore, the vibrational spectroscopy methods described here can also be used in fast quality checks of raw materials used in the pharmaceutical and flavour industries. First attempts to perform non-destructive NIR reflectance measurements on Umbelliferae fruits were made by Toxopeus and Bouwmeester [58]. They developed calibration models for the prediction of total essential oil and carvone contents in caraway fruits. Similar studies were done on caraway samples by another working group [59], resulting in a clearly higher prediction quality. Additionally, these scientists also established NIR calibrations for fennel fruits using numerous single plants with a wide range of essential oil contents (Figure 6) and different essential oil components such as estragole, fenchone and anethole [60]. Approximately 2 g of fruit were transferred into a standard reflectance cup without performing any clean-up procedures, and the NIR spectra were collected in the range from 1,100–2,500 nm. The NIR

method described was found to be a suitable approach for selecting high-quality single plants during the breeding process. The potential of NIR spectroscopy was also investigated for the determination of secondary metabolites in leaves from different Mentha species [61]. The results showed that simultaneous prediction of the oil content and its main terpenoid components menthol and menthone can be reliably achieved in dried peppermint (Mentha x piperita) (Table 2). For those components that usually occur in smaller amounts (for example menthofuran, isomenthone and pulegone), at least a semi-quantitative determination is possible. It has been found that the amounts of essential oil, menthol, menthone, isomenthone and 1,8-cineole can also be reliably predicted on fresh plant material. Thus, this NIR method can be applied as a useful tool for defining the optimal harvest time with respect to total essential oil and menthol content. NIR measurements performed on sage leaves (Salvia officinalis) [62] showed similar high prediction quality. As presented in Table 3, the individual essential oil and thujone contents can be analysed within a few seconds, not only in the dried herb, but also in fresh leaves. Since these two quality parameters have been found to be genetically determined, a fast and reliable selection of appropriate sage genotypes

Table 3. Range, mean and NIR correlation statistics for the amounts of essential oil (mL/100 g in the plant material) and thujone (g/100 g in the essential oil) in different sage genotypes. All NIR predictions are based on results of GC analyses of dried leaves.

Dried leaves

Fresh leaves

Essential oil

Thujone

Range

0.33–3.46

1.01–27.11

Mean

1.63

4.75

SECV

0.17

1.27



0.92

0.89

SEV

0.18

1.21

BIAS

0.02

0.25

SECV

0.21

2.01



0.88

0.79

SEV

0.24

1.79

BIAS

0.03

2.21

Abbreviations: GC, gas chromatography; NIR, near infrared; R², multivariable coefficient of determination; SEV, standard error of validation 8

Krüger and Schulz / Stewart Postharvest Review 2007, 4:4

Table 4. Application of Nnear infrared spectroscopy methods in the analysis of medicinal and aromatic plants.

Medicinal and aromatic plant sample

Analyte

Reference

Hop (Humulus lupulus)

Essential oil, myrcene, caryophyllene, humulene, farnesene, cohumulone

[68], [69]

Poppy (Papaver somniferum)

Total alkaloids, morphine, codeine, thebaine, papaverine, noscapine

[72]

Rosemary (Rosmarinus officinalis)

Carnosic acid, essential oil, terpenoid composition

[80], [81], [82]

Sage (Salvia officinalis)

Carnosic acid, essential oil, terpenoid composition

[70]

Marjoram (Origanum majorana)

Essential oil, terpenoid composition

[83]

Chamomile (Chamomilla recutita)

Essential oil, α-bisabolol

[84]

Fennel (Foeniculum vulgare)

Essential oil, fenchone, anethole

[80], [85], [86]

Caraway (Carum carvi)

Essential oil, carvone, limonene

[60], [80], [58]

Peppermint (Mentha piperita)

Essential oil, menthol, menthone and other terpenoids

[61]

Thyme (Thymus vulgaris)

Essential oil, thymol, carvacrol

[70]

Rooibos tea (Aspalathus linearis)

Aspalathin

[87], [88], [89]

Ginseng (Panax ginseng)

Ginsenosides Rg1, Rb1, Re

[90], [91]

Senna angustifolia

Sennosides A and B

[92]

Primrose (Primula veris)

3’,4’,5’ trimethoxyflavone

[93]

Echinacea species (E. pallida and E. angustifolia)

Echinacoside

[94]

Devil’s claw (Harpagophytum procumbens)

Harpagoside

[82]

Red pepper (Capsicum annuum)

Capsaicin

[95]

within breeding experiments is possible. The composition of marjoram oil and its occurrence in various marjoram cultivars (Origanum majorana) have been extensively studied [63–65]. It is known that during hydrodistillation cissabinene hydrate and cis-sabinene hydrate acetate are transferred on a bigger scale to artifacts such as terpinene-4-ol, αterpinene and γ-terpinene. In order to get a more authentic description of the phytochemical plant status, marjoram herb was carefully extracted with isooctane and subsequently analysed by GC; based on the obtained reference data an NIR calibration equation was successfully established [66]. The active components of rosemary leaves (Rosmarinus officinalis) [67], hop (Humulus lupulus) [68, 69], thyme (Thymus vulgaris) [70] and basil (Ocimum basilicum) [71] were determined in a similar way. Some applications of NIRS methods in analysis of medicinal and aromatic plants are listed in Table 4. IR spectroscopy was primarily used as a structure-elucidation technique; only limited application can be found in literature for characterisation of liquid samples such as fruit juices or

other plant extracts. Until recently, Raman spectroscopy was restricted mostly to academic research. Modern developments of the equipment now allow a broader application. Fourier transform (FT) infrared spectroscopy using a diamond composite attenuated total reflection (ATR) crystal and NIR-FT-Raman spectroscopy techniques were applied for the simultaneous identification and quantification of the most important alkaloids in poppy capsules [72]. Other examples for the use of ATR-IR and Raman spectroscopy in the field of medicinal and aromatic plants are: − the chemotaxonomic characterisation of essential oil plants (basil, chamomile, thyme, oregano) [73] − the identification of secondary metabolites in medicinal and spice plants (fennel, chamomile, curcuma) [74] by micro-spectroscopic mapping − the identification and quantification of harpagoside in secondary roots of Harpagophytum procumbens and its phytopharmaceutical products [75] − the determination of piperine in black pepper and green 9

Krüger and Schulz / Stewart Postharvest Review 2007, 4:4

− − −

whole pepper berries, as well as pepper oleoresins [76] the non-destructive analysis of single seeds of gene bank accessions belonging to various species of the Apiaceae family [77] the in situ identification of aspalathin and quantification of the dihydrochalcones in dried, green rooibos (Aspalathus linearis) [78] the observation of structural changes of polyacetylenes in American ginseng roots [79]

15

Krüger H, Schulz H, Steuer B and Zeiger B. Analytische Schnellmethoden zur Evaluierung ätherischer Öle in Genbankmaterial. Fachtagung “Arznei- und Gewürzpflanzen”, Tagungsband: 75–80, Gießen; 1998.

16

Lopez R, Aznar M, Cacho J and Ferreira V. Determination of minor and trace volatile compounds in wine by solid-phase extraction and gas chromatography with spectrometric detection. Journal of Chromatography A 2002: 966: 167– 177.

17

Krüger H. Separation of essential oil components from distillation water. Euro Cosmetics 2004: 11/12: 20–22.

18

Halkier BA and Gershenzon J. Biology and biochemistry of glucosinolates. Annual Review of Plant Biology 2006: 57 : 303–333. Boelens M, de Valois P and Wobben HJ. Volatile flavour compounds from onion. Journal of Agricultural and Food Chemistry 1971: 19: 984–991.

19

Conclusion There is a great inhomogeneity of active substances within and between the individual medicinal and aromatic plant species, even more strongly than that which exists in other cultivated species. The reason is that these plants are of a stronger wild character. This has consequences not only for breeding, but also for sampling and analysis. It is therefore important for future research activities to gain more exact knowledge regarding the distribution of active substances in plant parts, single plants and field parcels.

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