traceability in multi-ingredient botanicals by hptlc...
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Journal of Planar Chromatography 26 (2013) 3, 243–247 DOI: 10.1556/JPC.26.2013.3.7 2430933-4173/$ 20.00 © Akadémiai Kiadó, Budapest
SummaryA high-performance thin-layer chromatography (HPTLC) method
was developed for simple and rapid chemical analyses of multi-
ingredient botanicals. The method was based on comparison of the
fingerprint of the marketed multi-ingredient botanical with the fin-
gerprints of the extracts of its component plants and of a handmade
mixture of the constituent extracts in a ratio as similar as possible to
that of the market product, when labeled. Planar chromatography
analysis was completed by a densitometric scansion. The analyses
revealed a good correspondence between the market products and
the handmade mixture of standard extracts.
1 Introduction
For a long time, the utilization of a single chemical substance
was the basis of any development of medicinal drug and modern
pharmacology. In natural products, the analytical approach for
their validation graduated into the selection of one or few com-
pounds as either active constituents or “markers” for purposes of
identification and quality assessment. However, the last decay
evidenced a clear tendency for use of pharmaceutical multi-
ingredients in modern medicine, like those utilized for the treat-
ment of AIDS, cancer, hypertension, and viral infections. Even
more, the multi-ingredients presence became clear in the new
entries of the food sector, from nutraceuticals to functional
foods.
Evidences for the validity of the multi-ingredients approach can
be obtained by several studies on natural substances. Multiple
and multivalent pharmacological effects can be the consequence
of a complex chemical composition, like in echinacea, St. John’s
wort and garlic, or of a series of connected activity of multi-herb
products, like those marketed for treatment of dyspepsia and
motility-related disorders of gastrointestinal tract, where each
plant is intended for a specific treatment [1].
Recently, the complexity was increased by the massive intro-
duction on the market of products based on a lot of plant raw
materials, mainly derived from Traditional Medicines, includ-
ing, first for importance, the Traditional Chinese Medicine
(TCM). As a matter of fact, these medicines, derived from his-
torical references and empirical evidences, are based on multi-
ingredient preparations expressing the in toto activity of the
herbal drug mixture. On the other hand, each herbal drug con-
tains a myriad of different compounds, forming a complex
matrix of chemical interactions. Global market is an easy way
for everywhere potential diffusion of TCM medicines and other
traditional herbal drugs that, however, need adequate controls.
The analysis of a combination of herbal drugs constitutes an
overall difficult challenge.
In any analytical problem, the choice of the most suitable tool
must be first considered on the basis of its efficiency towards the
specific matter to be examined. For living organisms, due to
their complexity and variability, a holistic approach should be
applied not to lose any essential information. In this case, we
face an entire pattern of compounds, and the goal is to obtain a
complete visualization of all detectable constituents, not only
the markers [2–4].
High-performance thin-layer chromatography (HPTLC) has
been used as a useful tool for analysis of botanicals and multi-
ingredient products. However, so far the reported analyses are
based on the use of selected pure substances as standards or
markers [5, 6]. In this paper, another approach, based on HPTLC
fingerprint, is proposed.
A chromatographic fingerprint is the individual track represent-
ing, as near as possible, the mixture of organic substances pro-
duced by a living organism [7]. Fingerprint approach is a prod-
uct of metabolomic philosophy, the “study of as many small
molecules as possible” in an organic system. By the fingerprint
approach, it is possible to obtain a proper identification of the
plant material, but also determine and assert the limits of the
biological changes, without necessarily identifying or quantify-
ing a specific compound(s) [7, 8].
Traceability in Multi-Ingredient Botanicals by HPTLC Fingerprint Approach
Marcello Nicoletti*, Chiara Toniolo, Francesca Romana Gallo, Giuseppina Multari, and Giovanna Palazzino
Key Words
HPTLC Botanicals Multi-ingredient
M. Nicoletti and C. Toniolo, Quality Control Center, Department of Environmen-tal Biology, University Sapienza of Rome, P.le A. Moro 5 00185 Rome, Italy; andF.R. Gallo, G. Multari, and G. Palazzino, Department of Therapeutic Research andMedicines Evaluation, Istituto Superiore di Sanità, V. le Regina Elena 299, I-00161 Rome, Italy.E-mail: [email protected]
Traceability in Multi-Ingredient Botanicals by HPTLC Fingerprint Approach
244 Journal of Planar Chromatography 26 (2013) 3
Several methods (HPLC, LC–ESI–MS, PCR, etc.) have been
proposed to analyze botanicals by fingerprint approach, but usu-
ally they are expensive, not general and complicated [9–11].
HPTLC is able to generate a rapid, low-cost, and easily under-
standable chromatographic fingerprint in the form of a unique
sequence of peaks, nearly corresponding to the analyzed sample
in its chemical fullness [5]. In HPTLC tracks of the same
species, variations are generally quantitative, not qualitative.
Also HPTLC densitometric profiles were used.
In this study, we tested the HPTLC fingerprint approach on
identification and quality assessment of marketed multi-ingredi-
ent botanicals.
2 Experimental
2.1 Chemicals, Reagents, Materials, and Solutions
Methanol for analysis and HPLC grade solvents were purchased
from Sigma-Aldrich (Milan, Italy) and Carlo Erba (Milan,
Italy). Arbutin (AR, purity ≥96%) was purchased from Sigma-
Aldrich (Milan, Italy). Detailed information on the analyzed
samples, i.e., producers, production conditions, storage method,
etc., can be obtained by directly asking the authors. Extracts uti-
lized as mono-herbal standards were hydro-alcoholic 95°
extracts obtained from the market or by lab extraction of identi-
fied herbal raw materials (5 mg/10 mL); the last obtained
extracts were also used as references to confirm the identities of
the marketed ones. Several marketed multi-ingredient products
were analyzed in order to test the fingerprint approach. Market-
ed products 1–6 were obtained from the same Italian producer
(Herbal Farm Mario Caira, Villa Latina [FR], Central Italy),
selected on the basis of the high quality of its product and its
cool exsiccation method avoiding most of degradation effects.
Details concerning the producer and the production procedure
are available by asking the corresponding author.
2.2 Chromatographic Equipment
The HPTLC system (CAMAG, Muttenz, Switzerland) consisted
of (1) Linomat 5 sample applicator using 100 mL syringes and
connected to a nitrogen tank; (2) chamber ADC 2 containing
twin trough chamber 20 × 10 cm; (3) Immersion device III; (4)
TLC Plate Heater III; (5) TLC visualizer; and (6) TLC Scanner
3 linked to winCATS software.
2.3 Sample Preparation
The dried extracts of the analyzed samples were weighted and
dissolved in methanol (6 mg mL–1).
2.4 HPTLC Plates
Glass plates 20 cm × 10 cm (Merck, Darmstadt, Germany) with
glass-backed layers silica gel 60 F254 (2 μm thickness). Before use,
plates were prewashed with methanol and dried for 3 min at 100°C.
2.5 Sample Application
Filtered solutions were applied with nitrogen flow. The operat-
ing conditions were as follows: syringe delivery speed, 10 s μL–1
(100 nL s–1); injection volume, 2 μL; band width, 6 mm; dis-
tance from bottom, 15 mm.
2.6 Development
Before HPTLC analysis, HPTLC plates were pre-run using
MeOH, in order to avoid contaminants, and carefully dried.
Then the HPTLC plates were developed in the automatic and
reproducibly developing chamber ADC 2, saturated with the
same mobile phase, ethyl acetate–dichloromethane–acetic
acid–formic acid–water (100:25:10:10:11; v/v/v/v/v) for 20 min
at room temperature. The developing solvents (i.e., type of sol-
vents and ratios) were carefully optimized before the analyses.
The length of the chromatogram run was 80 mm from the point
of application.
2.7 Derivatization Reagents
The developed layers were allowed to dry in air for 5 min and
then derivatized with a selected solution, including anisaldehyde
(1.5 mL p-anisaldehyde, 2.5 mL H2SO4, 1 mL AcOH in 37 mL
EtOH) and/or Natural Product Reagent (NPR) (1 g diphenyl-
borinic acid aminoethylester in 200 mL of ethyl acetate), dried
in the open air and then dipped into Macrogol reagent (1 g poly-
ethylene glycol 400 in 20 mL of dichloromethane). Finally, the
plates were warmed for 5 min at 120°C before inspection.
2.8 Inspection
All treated plates were then inspected under UV light at 254 or
366 nm or under reflectance and transmission white light
(WRT), respectively, at a CAMAG TLC visualizer, before and
after derivatization.
2.9 Densitometric Analysis
For densitometric analysis, the scanner was set at 366 nm, after
a multi-wavelength scanning between 190 and 800 nm in the
absorption mode had been preliminarily tried. Minimum back-
ground compensation was performed on the x-axis during the
scanning [12]. The sources of radiation were deuterium and
tungsten lamps. The slit dimension was kept at 6.00 × 0.45 mm,
and the scanning speed used was 100 mm s–1.
2.10 Documentation
CAMAG DigiStore2 digital system with winCATS software
1.4.3 was used for the documentation of derivatized plates.
2.11 Stability
Sample solution of the extracts was prepared and stored at room
temperature for 3 days and then applied on the same HPTLC
plate and the chromatogram evaluated for additional band. Sim-
ilarly, band stability was checked by keeping the resolved peaks
and inspecting at intervals of 12, 24, and 49 h.
2.12 Validation
Overlapping of bands is a typical analytical challenge for com-
plex mixtures like multi-ingredient products. HPTLC allowed a
good separation and visualization of the constituents. Sample
solutions of the extracts were found to be stable at 4°C for at
least 1 month and for at least 3 days on the HPTLC plates.
Repeatability was determined by running a minimum of three
analyses. RF
values for main selected compounds varied
±0.02%. The effects of small changes in the mobile phase com-
position, mobile phase volume, and duration of saturation were
minute and reduced by the direct comparison. On the contrary,
the results were critically dependent on prewashing of HPTLC
plates with methanol.
3 Results and Discussion
In our analyses, the fingerprint of the marketed multi-ingredient
botanicals was compared with the fingerprints of the extracts of
the constituent plants and of a handmade mixture of the con-
stituent extracts in a ratio as similar as possible to that of the
market product, when labeled. In this way, it is possible for a
careful comparison of the fingerprints and a checking of the
used plant extracts. Owing to the large range of variability in the
herbal drug composition, it is crucial to obtain as many data as
possible, that means compare the fingerprints at different lights
and after several derivatizations. Thus, in product 1, reported in
Figures 1 and 2, the nettle extract presented a key spot with a
red fluorescent quenching at 366 nm after derivatization with
NPR, also confirmed in the densitometric scansion at the same
wavelength.
In Figure 2, the densitometric scansion of the plate at 366 nm
evidenced some quantitative differences between the commer-
cial product based on Bearberry (Arctostaphylos uva-ursi [L.]
Spreng) and Nettle (Urtica dioica L.) (Track 1) and the mix of
the two plants (Track 4), made in the lab. Both methods are use-
ful, direct visualization for an immediate check of the planar
chromatography and densitometric scansion to quantify the
results. By the second methodology, the quantitative differences
between the marketed product and the lab spiked one can be evi-
denced.
Several possible variations must be considered affecting the
extracts, like the herbal drug, ante- and post-harvesting and
extraction procedures, etc. Thus, in the analyzed marketed
multi-ingredient products, the absence of the typical red spots in
the layer front can be noticed. These spots are usually due to
chlorophylls and fat degradation products. However, in the ana-
lyzed products, extracts of raw plant materials were obtained by
the producer farm using a special exsiccation procedure at low
temperature by air fluxes, in order to minimize the degradation
effects. Other examples of similar HPTLC analyses are reported
in Figures 3–7.
In conclusion, some variabilities should be considered: (a) sev-
eral quantitative differences that must be accepted as a possible
result of variances in the raw materials, storage, and extraction
processes; (b) differences in the spots at high RFvalues absent in
the commercial product that could be attributed to lipophylic
constituents and chlorophylls degradation; (c) differences in the
relative presence of the various extracts.
Traceability in Multi-Ingredient Botanicals by HPTLC Fingerprint Approach
Journal of Planar Chromatography 26 (2013) 3 245
Figure 1
HPTLC visualization of marketed product 1 in comparison with con-stituent extracts. Derivatization: nothing; visualization: (a) UV 254 nm(b) 366 nm and (c) at WRT light; Tracks: 1. Commercial multi-ingredi-ent product 1 based on common nettle (Urtica dioica L.) and bear-berry (Arctostaphylos uva-ursi (L.) Spreng.), 2. A. uva-ursi leaves, 3. U. dioica aerial part, 4. Mix of A. uva-ursi and U. dioica.
Figure 2
HPTLC densitometric analyses of tracks 1–4 of Figure 1.
Figure 3
HPTLC analysis of marketed product 2 in comparison with con-stituent extracts. Derivatization: NPR; a. visualization: WRT; b. visu-alization: UV 366 nm; c. derivatization: NPR and anisaldehyde; visu-alization: WRT. Tracks: 1. Commercial multi-ingredient product 2based on helichrysum aerial part (Helichrysum italicum Roth), black-currant leaf (Ribes nigrum L.), lemon fruit (Citrus limon L.); 2.extracts of helichrysum (H. italicum Roth), 3. blackcurrant (Ribesnigrum L.) and 4. lemon (C. limon L.) reference plants, respectively;5. mixture of extracts 2–4.
The obtained results showed a general overlapping of the fin-
gerprint tracks in the original marketed product and in the mix-
ture made in the lab with the same constituent extracts of the
commercial product. The analysis can be also enriched by the
use of marker standards in comparison.
Furthermore, in each analysis, all the different images must be
considered and carefully examined at different conditions, since
some constituents were visible only in certain situations. An
important limit derives from the number of the utilized mixed
plants, that can complicate the mixture fingerprint, resulting in
difficulties to identify constituents devoid of clear or evident
spots.
The use of the fingerprint approach for identification of raw
materials has been already accepted and certified in some cases
[13–16]. The examples presented in this paper confirm the role
of HPTLC chromatographic fingerprint analysis in the quality
assessment of complex botanicals.
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Traceability in Multi-Ingredient Botanicals by HPTLC Fingerprint Approach
246 Journal of Planar Chromatography 26 (2013) 3
Figure 4
HPTLC analysis of marketed product 3 in comparison with con-stituent extracts. a. Visualization: UV 254 nm without derivatization;b. derivatization: NPR; visualization: UV 366 nm; c. derivatization:NPR and anisaldehyde; visualization: WRT. Tracks: 1. Commercialmulti-ingredient product 3 based on centaury aerial part (Centauriumerythraea Rafn), lemon balm leaves (Melissa officinalis L.), greaterburdock root (Arctium lappa L.); 2. extracts of centaury (C. erythraeaRafn), 3. lemon balm (M. officinalis L.), and 4. burdock (A. lappa L.),reference plants, respectively; 5, mixture of extracts of tracks 2–4.
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HPTLC analysis of marketed product 4 in comparison with con-stituent extracts. a. Visualization: UV 254 nm without derivatization;b. derivatization: NPR; visualization: UV 366 nm; c. derivatization:NPR and anisaldehyde; visualization: WRT. Tracks: 1. Commercialmulti-ingredient product 4 based on white willow bark (Salix alba L.),pot marigold flowers (Calendula officinalis L.), lesser calamint flower(Calamintha nepeta-savi L.); extracts of 2. white willow (S. alba L.), 3.pot marigold (C. officinalis L.), 4. lesser calamint (C. nepeta-savi L.),reference plants, respectively; 5, mixture of extracts of tracks 2–4.
Figure 6
HPTLC analysis of marketed product 5 in comparison with con-stituent extracts. a. Visualization: UV 366 nm without derivatization;b. derivatization: NPR; visualization: UV 366 nm; c. derivatization:NPR and anisaldehyde; visualization: WRT. Tracks: 1. Commercialmulti-ingredient product 5 based on white deadnettle aerial partLamium album L.), common nettle aerial part (Urtica dioica L.), potmarigold flower (Calendula officinalis L.); extracts of 2. white dead-nettle, (L. album L.), 3. common nettle (U. dioica L.), 4. pot marigold(C. officinalis L.), reference plants, respectively; 5, mixture of extractsof tracks 2–4.
Figure 7
HPTLC analysis of marketed product 6 in comparison with con-stituent extracts. a. Visualization: UV 366 nm without derivatization;b. derivatization: NPR and anisaldehyde; Visualization: UV 366 nm; c.derivatization: NPR and anisaldehyde; visualization: WRT. Tracks: 1.Commercial multi-ingredient product 6 based on butchers broom(Ruscus aculeatus L.) and bilberry (Vaccinium mirtyllus L.); 2.extracts of butchers broom (R. aculeatus L.), 3. bilberry (V. mirtyllusL.), reference plants, respectively; 4. mixture of extracts of tracks 2and 3; 5. arbutin.
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Ms received: June 15, 2012
Accepted: December 17, 2012
Traceability in Multi-Ingredient Botanicals by HPTLC Fingerprint Approach
Journal of Planar Chromatography 26 (2013) 3 247