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: marcello.nicoletti@uniroma1.it

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.

References

[1] H. Wagner, Revival of Pharmacognosy. Classical Botanical Phar-

macognosy. Satellite Symposium: Annual Meeting of the Ameri-

can Society of Pharmacognosy, Phoenix, AZ, 2004.

[2] M. Nicoletti, Nat. Prod. Commun. 6 (2011) 1003–1005.

[3] M. Nicoletti, Braz. J. Pharmacogn. 21 (2011) 818–823.

[4] E. Reich, A. Schibli, A High-Performance Thin-Layer Chromatog-

raphy for the Analysis of Medicinal Plants, Thieme Medical Pub-

lishers Inc., New York, 2006.

[5] Z.H. Song, Z.Z. Qian, C. Swamy Rumalla, T.J. Smillie, I. Khan,

J. Planar Chromatogr. 24 (2011) 312–315.

[6] P. Xie, S. Chen, Y.-Z. Liang, X. Wang, R. Tian, R. Upton, J. Chro-

matogr. A 1112 (2006) 171–180.

[7] Y. Zhang, S. Shujin, D. Yianye, W. Wenya, C. Huijuen, W. Jianhing,

G. Xiaojun, Intech Open Access Pub. Clinical Practise, Particular

Techniques and Special Issue. Cap. 10 (2011) 171–194.

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.

Figure 5

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.

[8] C.A. Pereira, J.H. Yariwake, F.M. Lanças, J.N. Wauters, M. Tits,

L. Angenot, Phytochem. Anal. 15 (2004) 241–248.

[9] F.R. Gallo, G. Multari, E. Federici, G. Palazzino, M. Giam-

benedetti, V. Petitto, F. Poli, M. Nicoletti, Nat. Prod. Res. 25 (2011)

1261–1270.

[10] S.-K. Wong, S.-K. Tsui, S.-Y. Kwan, X.-L. Su, R.-C. Lin, L.-M. Tang,

J.-K. Chen, J. Food Drug Anal. 12 (2004) 110–114.

[11] L. Mattioli, F. Cangi, C, Ghiara, M. Burico, A. Maidecchi,

E. Bianchi, E. Ragazzi, L. Bellotto, R. Seraglia, P. Traldi, Meta-

bolomics 7 (2011) 437–445.

[12] P. Del Serrone, L. Attorri, G. Palazzino, Nat. Prod. Res. 21 (2007)

1099–1103.

[13] WHO, World Health Organization, Quality Control Methods for

Medicinal Plant Materials. WHO Library Cataloguing, Geneva, 2011.

[14] TLC Atlas of Chinese Crude Drugs in Pharmacopoeia of the Peo-

ple’s Republic of China Chinese Pharmacopoeia Commission,

People’s Medical Publishing House, Beijing, 2009.

[15] The United States Pharmacopoeia, 31st Edition. The National For-

mulary, 26th edition. The United States Pharmacopoeial Conven-

tion, Rockville, MD.

[16] A. Gautan, S.J. Kashyap, P.K. Sharma, V.R. Garg, S. Vish,

N. Kumar, Pharm. Lett. 2 (2010) 302–315.

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