**2. An example of differentiation of** *Baccharis* **L. species belonging to sect – Caulopterae DC. (Asteraceae) using UV/Visible spectrophotometry data and multivariate analysis**

*Baccharis* L. is an exclusive American genus comprising approximately 500 species. Its distribution area covers the whole of South America and continues northwards up to the south of USA including the Atlantic coast up to Massachusetts, although its presence is greater in the intertropical and subtropical zones (Cuatrecasas, 1967; Müller, 2006). Several

Quality Control of Herbal Medicines with Spectrophotometry and Chemometric Techniques

medicinal purposes due to their similar morphotypes as they have alate stem.

**2.1 Material and methods** 

filter (Lonni et al*.*, 2003, 2005).

not be characterized by the previous method.

(Table 1).

France).

France).

– Application to *Baccharis* L. Species Belonging to Sect – Caulopterae DC. (Asteraceae) 405

et al*.*, 2003; Gene et al., 1992, 1996; Lapa et al., 1992; Palacios et al., 1983; Stoicke & Leng-Peschlow, 1987). Also the high frequency of errors committed during the collection of these species for medicinal purposes is understandable due to the coexistence of these entities in certain habitats. Hence, considering that numerical methods have been shown to be useful for multi-component metabolic classification studies, we decided to carry out population studies including 53 samples of these nine sect. Caulopterae species, combining their spectrophotometric profiles with multivariate analysis. These nine species belonging to sect. Caulopterae are: *Baccharis articulata* (Lam.) Pers., *B. crispa* Spreng., *B. gaudichaudiana* DC., *B. microcephala* (Less.) DC., *B. phyteumoides* (Less.) DC., *B. penningtonii* Heering., *B. sagittalis*  (Less.) DC., *B. triangularis* Hauman and *Baccharis trimera* (Less) DC. Of these nine species, only three are official and its use is permitted but all are indiscriminately collected for

Fifty three samples of nine *Baccharis* species were collected from wild materials in different locations in Argentina. All samples were botanically identified by our group and voucher specimens were deposited at the herbarium of the National University of Rosario, Argentina

The aerial parts of the dried plants (5 g) were macerated (24 h, 3x) with absolute ethyl alcohol. The ethanolic extract was filtered and concentrated in a rotary evaporator at a temperature lower than 100 ºC. Thirty mg of dry extract were mix in 3 ml dichloromethane (DCM) and left for 1 h and then filtered with common filter paper. A dilution of 50 µl of this solution in 950 µl of methanol was prepared and filtered twice with a 0.45 µm Millipore

Spectrophotometric analyses were carried out using a Biochrom Model Libra S12 UV/Visible Spectrophotometer, equipped with tungsten halogen and deuterium arc light

TLC analyses were carried out using silica gel 60 F254, Merck; mobile phase, DCM: Hexane: MeOH (4:2:1). Chromatograms were evaluated under UV light at 254 and 365 nm to detect the presence of flavonoids. TLC was additionally sprayed with a diphenylborinic acid ethanolamine/polyethylene glycol reagent. Apigenin, chlorogenic acid, genkwanin, luteolin, quercetin and rutin were used as markers (purchased from Extrasynthèse,

HPLC analyses were carried out using a Spectra Physics Model SP8800 ternary pump chromatograph with Spectra 100 UV/Visible detector, having as chromatographic conditions, methanol eluent, 1 ml min-1 flow, Luna C18 phenomenex (250 x 4.6 mm, 5 µm particle size). The injection volume was 100 µl and elution was monitored al 254 nm. Apigenin, genkwanin and luteolin were used as markers (purchased from Extrasynthèse,

TLC and HPLC analysis were applied in order to complement the studies carried out by PCA of the spectrophotometric data and to find potential markers of the species that could

sources with a single solid state silicon photodiode detector, and operating software.

Fig. 3. Two-dimensional model derived from PCA of 700 quantitative variables of 10 *Baccharis* populations. Ba, *B. articulata*; Bt, *B. trimera*. BA, Buenos Aires; COR, Corrientes; ER, Entre Rios; SF, Santa Fe populations. Numbers indicate when there is more than one population of the same species.

authors have contributed to the infrageneric classification of *Baccharis* in general and regional floras (Ariza Espinar, 1973; Baker, 1882-1884; Barroso, 1976; Cuatrecasas, 1967; De Candolle, 1836; Giuliano, 2001; Heering, 1904; Lessing, 1831; Weddell, 1855-1856) and it was De Candolle (1836) who was the first to subdivide the genus in eight sections, mainly based on leaf morphology. More recently, Giuliano (2001) grouped 96 Argentine *Baccharis* species into 15 sections, among which the sect. Caulopterae DC. is characterized by the presence of species with alate stems. Two of nine Argentine species from this sect., i.e., *Baccharis articulata* (Lam.) Pers. and *Baccharis crispa* Spreng. are included in the National Argentine Pharmacopeia Ed. VI (1978), and a third one, *Baccharis trimera* (Less.) DC. in the Brazilian Pharmacopeia Ed. IV (2002) with the common name of "Carquejas". The nine species of this sect. are traditionally used in infusions or decoctions, as hepatic, colagogue, diuretic, ulcer healing and external antiseptics. They are also used in herbal remedies and phytotherapy and in the preparation of spirits and soft drinks (Correa, 1985; Gupta, 1995; Hieronymus, 1882; Martínez Crovetto, 1981; Sorarú & Bandoni, 1978; Toursarkissian, 1980). Beneficial effects of these species can be attributed in part to their high content of flavonoids. The chemistry of the flavonoids is predictive of their free radical scavenging activity, which confers the antioxidant activity (Harborne & Williams, 1992; Rice-Evans et al., 1995).

Currently, the morphoanatomical studies of these species in sect. Caulopterae only provide incomplete information which makes it difficult to differentiate each one properly in the non-flowering condition when the size of capitulum of each one varies (Giuliano, 2000; Müller, 2006). This fact has lead to the misuse of the same common name for botanically diverse species, which surely have different chemical compositions and therefore different pharmacological properties (Abdel-Malek et al., 1996; Desmarchelier et al*.*, 1997; De Oliveira et al*.*, 2003; Gene et al., 1992, 1996; Lapa et al., 1992; Palacios et al., 1983; Stoicke & Leng-Peschlow, 1987). Also the high frequency of errors committed during the collection of these species for medicinal purposes is understandable due to the coexistence of these entities in certain habitats. Hence, considering that numerical methods have been shown to be useful for multi-component metabolic classification studies, we decided to carry out population studies including 53 samples of these nine sect. Caulopterae species, combining their spectrophotometric profiles with multivariate analysis. These nine species belonging to sect. Caulopterae are: *Baccharis articulata* (Lam.) Pers., *B. crispa* Spreng., *B. gaudichaudiana* DC., *B. microcephala* (Less.) DC., *B. phyteumoides* (Less.) DC., *B. penningtonii* Heering., *B. sagittalis*  (Less.) DC., *B. triangularis* Hauman and *Baccharis trimera* (Less) DC. Of these nine species, only three are official and its use is permitted but all are indiscriminately collected for medicinal purposes due to their similar morphotypes as they have alate stem.
