Phamacognostic Evaluation of Cissampelos Sympodialis Eichl Leaves

South African Journal of Botany 93 (2014) 70–78

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South African Journal of Botany

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Phamacognostic evaluation of Cissampelos sympodialis Eichl leaves

A.C. Cavalcanti a, A.N.P. Gomes b,N.M.Portob, M.F. Agra b, T.F.A.L. Moura a, E.J. Oliveira a,b,⁎ a Campus Universitário Lagoa Nova, Universidade Federal do Rio Grande do Norte, Programa de Pós-graduação em Desenvolvimento e Inovação Tecnológica em Medicamentos, CEP 59078-970 Natal, Rio Grande do Norte, Brazil b Cidade Universitária, Universidade Federal da Paraíba, Centro de Ciências da Saúde, Programa de Pós-graduação em Produtos Naturais e Sintéticos Bioativos, CEP 58051-900 João Pessoa, Paraíba, Brazil article info abstract

Article history: Background: Cissampelos sympodialis Eichl. (Menispermaceae) is used in folk medicine for the treatment of Received 27 January 2014 respiratory conditions such as bronchitis and asthma. The aqueous fraction of the hydroalcoholic extract of its Received in revised form 18 March 2014 leaves has shown immunomodulatory, bronchodilator and anti-inflammatory activities. However, there is no Accepted 22 March 2014 data available on the pharmacognostic quality control of its leaves. Available online xxxx Objective: The aim of the present study was to characterize the drug material obtained from the leaves of C. sympodialis, with the determination of WHO recommended assays, histochemistry of leaf sections and HPLC Edited by I Vermaak determination of its main bioactive alkaloid, warifteine. Keywords: Materials and methods: Three batches of leaves were collected and histochemical and phytochemical analyses Cissampelos were performed to screen for alkaloids, phenolics, terpenoids, lipids, polysacharides and saponins. Pharmacope- HPLC ial assays for the plant material were also conducted and the bisbenzylisoquinoline alkaloid warifteine was Medicinal plant quantified by HPLC analysis. Menispermaceae Results: The histochemistry of leaf sections from C. sympodialis revealed the presence of lipids, polysaccharides, Leaf analysis phenolic compounds and alkaloids, mainly as part of idioblast secretions. These results were confirmed by Quality control general phytochemical screening reactions conducted with the leaf hydroalcoholic extract (presence of alkaloids, Warifteine steroids, tannins, flavonoids and saponins). General pharmacopeial tests conducted with the leaves were within accepted standards. The warifteine content was similar for the three batches tested (0.004–0.006%). Conclusion: The physicochemical parameters and the morphological results presented in this paper can be used as basis for the preparation of a monograph on C. sympodialis leaves. © 2014 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction of herbal medicines, such as temperature, light exposure, water avail- ability, nutrients, period and time of collection, method of collecting, The World Health Organization (WHO) defines “herbal medicine” drying, packing, storage and transportation of raw material, age and as a plant-derived material or preparation with therapeutic or other part of the plant collected (Calixto, 2000). For this reason it is of utmost human health benefits which contains either raw or processed ingredi- importance to have raw materials with a consistent and reproducible ents from one or more plants (Zhang et al., 2012). The use of medicinal quality standard. plants has a long history throughout the world and herbal preparations, Cissampelos sympodialis, belongs to the Menispermaceae family, and including herbal extracts, can be found in the pharmacopeias of numer- is popularly known in Brazil as “milona”, “jarrinha”, “orelha-de-onça” ous countries (Huie, 2002; Nascimento et al., 2005). However, for medic- and “abuteira”. It occurs in many Brazilian states such as Paraíba, inal plants that do not appear in official codes, studies must be conducted Ceará, Alagoas and Bahia. Its medicinal use was confirmed by many re- to establish parameters to ensure their quality, according to technical ports. In Paraiba an aqueous infusion of the leaves was used for several and scientific principles (Barni et al., 2009; Shinde et al., 2009; Braz different respiratory diseases, but mostly asthma (Barbosa-Filho et al., et al., 2012). Quality control of herbal medicines aims to ensure their 1997; Agra and Barbosa-Filho, 2007). The study of C. sympodialis Eichl. consistency, safety and efficacy (Li et al., 2008). as potential pharmaceutical material should be encouraged if taken The source and quality of raw materials play a pivotal role in into account its abundant natural occurrence (Barbosa-Filho et al., guaranteeing the quality and stability of herbal preparations. Many 1997; Porto et al., 2008), its frequent use in folk medicine (Correa, factors can affect the quality and consequently the therapeutic value 1984; Barbosa-Filho et al., 1997; Alexandre-Moreira et al., 2003; Bezerra-Santos et al., 2006; Corrêa et al., 2008), the confirmation of the therapeutic potential in preclinical studies (Thomas et al., 1997a; ⁎ Corresponding author at: Centro de Biotecnologia, UFPB, Campus I, Cidade Universitária, CEP: 58051-970, João Pessoa, PB. Tel.: +55 83 3216 7371. Freitas et al., 2000; Bezerra-Santos et al., 2004, 2005, 2006, 2012; E-mail address: [email protected] (E.J. Oliveira). Barbosa-Filho et al., 2006; Cerqueira-Lima et al., 2010) and the

http://dx.doi.org/10.1016/j.sajb.2014.03.014 0254-6299/© 2014 SAAB. Published by Elsevier B.V. All rights reserved. A.C. Cavalcanti et al. / South African Journal of Botany 93 (2014) 70–78 71 knowledge of its chemical composition (Barbosa-Filho et al., 1997, 2.4. Preliminary phytochemical screening of C. sympodialis 2000; Marinho et al., 2012, 2013; Cavalcanti et al., 2013). Despite the preclinical pharmacological results, there is a lack of 2.4.1. Preparation of extracts specifications for the quality control of C. sympodialis raw material, Fifty grams of powdered leaves were extracted with 100 ml of etha- which is a pre-requisite for the production and registration of nol:water (80:20, v/v) by maceration for 24 h. The extract was filtered phytomedicines (Brasil, 2010). Thus, the aim of the present study was and the filtrate was evaporated to dryness under reduced pressure at to obtain and characterize the medicinal chemical compounds obtained 40 °C with a rotary evaporator. from the leaves of C. sympodialis, with the determination of parameters for quality control, including the determination in the levels of its active 2.4.2. Assays for specific metabolites principle/chemical marker warifteine (a bisbenzylisoquinoline alkaloid) by HPLC (Fig. 1). 2.4.2.1. Determination of the presence/absence of tannins. Approximately 200 mg of extract was diluted with water and added at three concentrations (25%; 50% and 100%) to 2% ferric chloride solution. Tannins give a 2. Materials and methods blackish blue or blackish green color in the presence of ferric chloride (Mace and Howell, 1974). Tannins were furthermore assayed by adding 2.1. Drugs and reagents approximately 200 mg of the extract to gelatin 0.5% at the same three concentrations and the presence of tannins was confirmed by gelatin Triethylamine and methanol were from (Tedia®), Ultrapure water, precipitate formation (Evans, 1989). was used for HPLC and was obtained using Purelab Option-Q (Elga®). Other reagents and solutions used for extraction, and histochemical 2.4.2.2. Determination of the presence/absence of flavonoids. Approxi- and phytochemistry analyses were reagent-grade. Warifteine was mately 200 mg of extract was dissolved in methanol (50%, 1–2ml)by isolated using a standard alkaloid acid–base extraction procedure that heating. Then, magnesium ribbon and 5–6 drops of concentrated hydro- used the ethanolic extract of the roots of C. sympodiallis as starting chloric acid were added. The solution turns red when flavonoids are material (Mukherjee and Keifer, 2003)followedbypurification and present. Flavonoid determination was also conducted using the boric identification (Marinho et al., 2012). acid complexation method where 5 drops of acetone were added to the extract followed by the addition of a few crystals of oxalic and boric acids. The solution was dried and 10 ml of ethyl ether was 2.2. Plant material added. The presence of fluorescence was investigated using an UV lamp at 365 nm (Harborne, 2005). Three batches of C. sympodialis leaves (approximately 4 kg of fresh material each) were collected from pooled specimens (Nov 2010, Jan 2.4.2.3. Determination of the presence/absence of steroid. Approximately 2011, and Jan 2012). The plant was cultivated at the botanical garden 200 mg of extract was dissolved in equal volumes of acetic anhydride at the Federal University of Paraiba (7°08′27,84″S 34°50′47,92″W). and chloroform. The mixture was transferred to a dry test tube and The botanical identification was made by comparison with the voucher concentrated sulfuric acid was added at the bottom of the tube. The specimen deposited at the Herbarium Prof Dr Lauro Pires Xavier, under presence of steroids in the Lieberman–Burchard's reaction is evidenced number Agra 1456. The batches of collected leaves were labeled as “1”, by the formation of a reddish brown or violet brown ring at the interface “2” and “3” according to increasing order of collection date and then of the two liquids (Raaman, 2006). dried at 40 °C, powdered in a knife mill and stored where it was protected from light and moisture. 2.4.2.4. Determination of the presence/absence of alkaloids. The alkaloids were extracted by refluxing 500 mg of extract with sufficient amount of water for about 2 h. The extract was concentrated on rotatory evapo- 2.3. Histochemical analysis rator, basified with about 1 ml of sodium hydroxide 1% and was extracted with 6 ml of chloroform (three times). An aliquot of 1 ml of the Transversal sections (leaf blade and petiole) and longitudinal chloroform phase was transferred to four test tubes for different test (petiole) were made in adult leaves by hand with a razor blade. Subse- reagents which include a few drops of Bouchadart (Raaman, 2006), quently, the sections were submitted to different dyes/reagents as spec- Mayer (Evans, 1989), Dragendorff (Waldi, 1965) and tungstic acid ified in Table 1. Control sections (unstained) were analyzed in parallel. (Raaman, 2006) reagents. Formation of an orange precipitate was For the observation of leaf powder, the dried leaf blade was powdered taken as a positive result for alkaloids. and diaphanized with 1% sodium hypochlorite (Franklin, 1945). The material was then washed with distilled water containing 50% safranin 2.4.2.5. Determination of the presence/absence of saponins. Approximately and mounted on 50% glycerin glass slides. Imaging was made with a 50 mg of extract was dissolved in 10 ml of water and was shaken vigor- Leica DM750 photomicroscope, equipped with an ICC50HD image ously to froth. After standing 10 min, the persistence of foam indicates capture system and the Qwin software. the presence of saponins (Kokate, 1999).

2.4.3. Determination of the moisture content Approximately 3 g of the prepared air-dried material was accurately weighed and placed in a previously dried and tared ﬂat weighing bottle. The sample was dried in an oven at 100–105 °C for 5 h until two consec- utive weights did not differ by more than 5 mg. The loss of weight expressed as percentage of air-dried material was calculated and the experiment was done in triplicate for each batch of leaves (WHO, 1998; Brasil, 2010).

2.4.4. Determination of total ash content Approximately 3 g of the ground air-dried material, accurately Fig. 1. Warifteine, a bisbenzylisoquinoline alkaloid and biomarker of C. sympodialis. weighed, was placed in a previously ignited and tared crucible of silica. 72 A.C. Cavalcanti et al. / South African Journal of Botany 93 (2014) 70–78

Table 1 Staining methods used for detection of the main classes of secondary metabolites in sections of C. sympodialis leaves.

Tests used Compound Metabolite group Reference

Sudam III, Sudam IV, Sudam black B Lipids Total lipids Johansen (1940) Schulze's solution (chlor-zinc-iodide) Polysaccharides Starch and cellulose Jensen (1962) Lugol Polysaccharides Starch Jensen (1962) Iron (III) chloride Phenolics General phenolic compounds Johansen (1940) Potassium dichromate Phenolics General phenolic compounds Gabe (1968) Vanillin–HCl Phenolics Tannins Mace and Howell (1974) Phloroglucinol–HCl Phenolics Lignin Johansen (1940) Wagner reagent Alkaloids Alkaloids Furr and Mahlberg (1981) Dittman reagent Alkaloids Alkaloids Furr and Mahlberg (1981) 2,4-Dinitrophenylhydrazine Terpenoids Terpenoids Ganter and Jollés (1969)

The material was distributed in an even layer and ignited by gradually 2.4.8. Warifteine content increasing the heat to 500–600 °C until the resulting residue was The concentration of warifteine in the hydroalcoholic extract was white, indicating the absence of organic matter. A gradient of tempera- determined using an HPLC method described in a recent study by ture with 200 °C for 30 min, 400 °C for 60 min and 600 °C for 90 min was Marinho et al. (2012). Samples were prepared at a concentration of used. The crucible was cooled in a desiccator for 30 min and weighed. 15 mg/ml of extract in 0.01% triethanolamine:methanol (40:60, v/v). The content of total ash was calculated as percentage of air-dried mate- The solution was filtered through nylon membrane (0.45 μm) before rial and the experiment was done in triplicate (WHO, 1998; Brasil, HPLC analysis was done in triplicate. Calibration curves with warifteine 2010). reference standard were prepared in the following concentrations: 2, 3, 5, 10, and 20 μg/ml for quantifying this marker in the samples. The warifteine content was calculated in terms of percentage in relation to 2.4.5. Determination of ethanol extractable content dry leaves. Approximately 3 g of coarsely powdered air-dried material, accu- fl rately weighed, was placed in a glass-stoppered conical ask. The 3. Results and discussion plant material was macerated with 100 ml of ethanol for 6 h, shaking fi frequently, and was then allowed to stand for 18 h. The extract was l- Vernacular names of plants vary in different regions of the country, fi tered rapidly taking care not to lose any solvent, and 25 ml of the ltrate which can cause serious problems in the use of medicinal plants fl was transferred to a tared at-bottomed dish and evaporated to dryness (Shinde et al., 2009) and can even lead to adulteration (Capasso et al., on a water-bath. It was dried (in an oven with circulation air) at 105 °C 2000; Zhang et al., 2012). The source and quality of raw materials play for 6 h., cooled in a desiccator for 30 min and weighed without delay. a pivotal role in guaranteeing the quality and stability of herbal prepara- The content of extractable matter was calculated in mg per ml of liquid tions (Calixto, 2000). Therefore botanical and chemical analyses of plant extract and the experiment was done in triplicate for each batch of species are of paramount importance for the identification and quality leaves (WHO, 1998; Brasil, 2010). control of herbal drugs (Brasil, 2010; Felix-Silva et al., 2012). A recent study (Marinho et al., 2012) indicated that the lowest alka- 2.4.6. Swelling index loid content in the leaves of C. sympodialis is found during fruit ripening, Approximately one gram of plant material, accurately weighed was which coincides with the appearance of the alkaloids in the fruits. For placed in a glass-stoppered measuring cylinder and was shaken repeat- this reason we conducted our study with plant material collected before fl edly every 10 min during 1 h. The volume of the mixture (in ml) was owering. measured after 3 h. The experiment was done in triplicate for each batch of leaves (Brasil, 2010). 3.1. Histochemical analysis

The main classes of metabolites identified in sections of leaves of 2.4.7. Particle size C. sympodialis by staining with specific reagents, after histochemical Approximately 25 g of leaves were subjected to sequential selected analysis, are summarized in Table 2. Secreting structures are reported sieves (1; 0.850; 0.710; 0.500; 0.425; 0.350; 0.297; 0.250; 0.149; for several genera of Menispermaceae such as: Cyclae, Diploclisia, 0.074 mm) by shaking for 30 min. The plant material retained on each Stephania and Cissampelos (Metcalfe and Chalk, 1957). These structures sieve was weighed and the powder was classified according to particle can present themselves in isolation constituting idioblasts, or forming size. multicellular structures, such as ducts or channels (Castro and Machado,

Table 2 Compounds identiﬁed in the leaves of C. sympodialis with different histochemical tests.

Compound Test Observed color Result

Lipids Sudam IV Red ++ Cutin Sudam black B Blue to black ++ Polysaccharides Lugol Bluish-black ++ Polysaccharides Schulze's solution (Chlor-zinc-iodide) Dark-brown + Phenolic compounds Iron (III) chloride Black + Phenolic compounds Potassium dichromate Reddish-brown + Phenolic compounds Vanillin–HCl Red − Lignin (cutin) Phloroglucinol–HCl Pink to red ++ Alkaloids Wagner reagent Reddish brown ++ Alkaloids Dittiman reagent Reddish brown ++ Terpenoids 2,4-Dinitrophenylhydrazine Orange −

Note: (−) absent; (+) present; number of signs indicate the intensity of the reaction. A.C. Cavalcanti et al. / South African Journal of Botany 93 (2014) 70–78 73

2006). In C. sympodialis the secreted material is stored in idioblasts of The phloroglucinol–HCl reagent revealed the presence of lignin in vascular tissues in the cortical parenchyma of the midrib and petiole. the vascular tissue, where these compounds are usually present However, in the other species of the genus, such as Cissampelos (Fig. 2E). It could also be seen as a continuous schlerenchymatic ring fasciculata Benth (Metcalfe and Chalk, 1957), Cissampelos hirta Klotzsch in the median portion of the midrib and petiole of C. sympodialis, and Cissampelos mucronata A. Rich (Wet et al., 2002) the secreted mate- which was previously described by Porto et al. (2008). Analysis of the rial was observed in secretory cavities, which were not observed in the powered leaves of C. sympodialis has a light brown color with character- leaves of C. sympodialis. istic structures from leaf epidermis, like stomata and epidermal tissue The reactions with Sudan IV and Sudan black B were positive, indicat- (Fig. 2F). ing lipophilic substances framing a thin cuticle in the leaves (Fig. 2A). The content of idioblasts reacted positive for Dittman and Wagner Idioblasts with calcium oxalate raphides occur on the interior of the reagents, with a reddish-brown color indicating the presence of alka- cortical parenchyma cells (Fig. 2B). Starch granules are frequent in loids (Fig. 3 A–C). The presence of alkaloids is a chemotaxonomic parenchymal cells of the rib and medular region of petiole as well as in feature of the Menispermaceae, allowing the separation of genera the palisade parenchyma of the mesophyll (Fig. 2C–D). according to the structural class of alkaloids being biosynthesized

Fig. 2. Photomicrographs of leaves of C. sympodialis, in cross-sections, after histochemical tests. A. Sudan IV reaction emphasizing the cuticle (cutin) of petiole; B. detail of petiole showing idioblasts with raphides of calcium oxalate; C. medullar region of the petiole stained black with Lugol, indicating the presence of starch (polysaccharides); D. detail of starch grains (polysaccharides) in medullar region of petiole; E. detail of vascular bundle stained red and red-wine (arrowhead) with phoroglucinol–HCl, indicating the presence of lignin; F. powdered leaves with segments of epidermal tissue and stomata (arrowhead). (cut: cuticle; scl: schlerenchyma; phl: phloem; xl: xylem; rp: raphides; par: parenchyma; sg: starch grains; st: stomata; vc: vascular cambium.) 74 A.C. Cavalcanti et al. / South African Journal of Botany 93 (2014) 70–78

(Menachery, 1996; Barbosa-Filho et al., 2000). These results are in The reaction of tissues to concentrated sulfuric acid for the detection agreement with chemical studies already conducted with this species of saponins and/or sesquiterpene lactones in the leaves was considered and allowed the location of the production and storing sites of these nonconclusive. Some parenchymatic cells around the vascular bundles compounds in the leaf blade. showed the brown color pattern as expected for these compounds. The reactions for phenolic compounds were positive for both iron The reaction to terpenes and tannins was negative. (III) chloride and potassium dichromate reagents (Fig. 3D–F). Idioblasts The metabolites displaying the most positively intense histo- presented a dark color in the parenchymal cells of the petiole and in the chemistry reactions in the leaf sections of C. sympodialis were: alka- palisade parenchyma of the mesophyll. These results are consistent loids, phenolic compounds, acid polysaccharides and total lipids. with previous descriptions for a related species, C. parreira L., for These results conﬁrm the complexity of idioblast secretions, and which this class of metabolites was also detected in its hydroalcoholic indicate fundamental tissues as the sites for synthesis and storage extract (Samanta et al., 2012). of these compounds.

Fig. 3. Photomicrographs of cross and longitudinal sections of leaves of C. sympodialis, after histochemical tests. A. Cross section of petiole showing idioblasts stained dark brown with Dittman's reagent as positive for alkaloids; B. detail of idioblasts in phloem of petiole as positive reaction for alkaloids; C. longitudinal section stained dark brown as positive reaction for alkaloids; D. idioblasts with phenolic compounds stained dark-brown under the epidermis of petiole; E. cross section of petiole stained dark-brown color with potassium dichromate as positive reaction for phenolic compounds; F. detail of a idioblast with phenolic compounds stained brown. (cut: cuticle; scl: schlerenchyma; phl: phloem; xl: xylem; rp: raphides; par: parenchyma; sg: starch grains; st: stomata; vc: vascular cambium.) A.C. Cavalcanti et al. / South African Journal of Botany 93 (2014) 70–78 75

Table 3 Phytochemical screening of three batches of leaves of C. sympodialis.

Compound Test Results Font

Batch I Batch II Batch III

Alkaloids Bouchardat + + + Raaman (2006) Mayer ++ + ++ Evans (1989) Dragendorff ++ ++ +++ Waldi (1965) Silic–tungistic acid + − ++ Raaman (2006) Steroids Steroids ++ ++ ++ Raaman (2006) Tannins Gelatin (25%) −− − Evans (1989) Gelatin (50%) − ++ Evans (1989) Gelatin (100%) + ++ + Evans (1989)

FeCl3 (25%) −− + Mace and Howell (1974)

FeCl3 (50%) + ++ ++ Mace and Howell (1974)

FeCl3 (100%) ++ ++ ++ Mace and Howell (1974) Flavonoids Magnesium + ++ ++ Harborne (2005) Fluorescence ++ ++ ++ Harborne (2005) Saponins Foam − ++ Kokate (1999)

The symbol (−) means absence and (+) means presence of substance class. Numbers of symbols are directly related to the intensity of the reaction.

3.2. Preliminary phytochemical screening of C. sympodialis specified range (Table 4). Preliminary operations employed in the leaves were therefore effective in reducing the moisture content to The phytochemical screening of C. sympodialis leaves indicated the acceptable levels (Barni et al., 2009). presence of the following secondary metabolites: alkaloids, which were mostly reported in this plant (Cavalcanti et al., 2013), steroids, 3.4. Determination of total ash content flavonoids, cumarins and saponins (Table 3). These data show groups of substances that can be used in the characterization of the species The measurement of ashes is intended to establish the quantity of raw material, such as alkaloids, and the absence of a positive reaction non-volatile inorganic content. The ash content comprises both the for some substances may be useful to differentiate related species physiological ashes, derived from the plant tissues, as well as non- (Daleffi-Zocoler et al., 2006; Hubinger et al., 2009). The results from physiological ashes (Farias, 2003; Migliato et al., 2007). Elevated ash the histochemistry analysis were corroborated (with the exception of contents in aerial material of interest (leaves, inflorescences and saponins for which histochemistry results were not conclusive), and flowers) may indicate external material adhered to the surface of the confirmed the presence of phenolic compounds and alkaloids in the plant, such as sand and silica (Fonseca et al., 2010). hydroalcoholic leaf extract. The leaves of C. sympodialis have a considerable percentage of ash (Table 4), but the values are within the specified range of 5–15% 3.3. Determination of moisture content (WHO, 1998; Brasil, 2010) for medicinal plant material.

Moisture content is a parameter that can interfere considerably in 3.5. Determination of ethanol extractable content and swelling index the stability of the active constituents present in the plant, since excessive water in the plant's raw material favors the action of enzymes The content of extractable substances determines the amount of whose activity may lead to the degradation of chemical constituents, substances with a possibility of being extracted by a specified solvent, as well as enabling the development of fungi and bacteria (Barni et al., in this case ethanol (WHO, 1998). The extraction solvent must take 2009; Hubinger et al., 2009). Specifications for the maximum allowable into account both the polarity of the target constituents (Michelin values for loss on drying are an important information from a techno- et al., 2010), as well as the water content regarding the risk of microbi- logical point of view and also serve as parameter for quality control of ological contamination (Migliato et al., 2007). The results between the plant material (Migliato et al., 2007). batches show some similarity between the content of extractable The specification for moisture content established in several substances, which ranged between 2.51 and 3.59 mg/ml of extract pharmacopeias, including the Brazilian pharmacopeia, is in the range (Table 4). The absence of reference data for extractable content of of 8–14% in dry plant material (WHO, 1998; Brasil, 2010), with few Cissampelos species makes this data informative only (as well as swell- exceptions in some monographs (Hubinger et al., 2009; Fonseca et al., ing index, that range between 1.67 to 2.33 ml), but relevant for quality 2010). Although there was some variation between the batches of control of the plant raw material, along with preliminary botanical leaves evaluated, the moisture content of each of them was within the and phytochemical data (Daleffi-Zocoler et al., 2006).

Table 4 Moisture content, total ash content and ethanol extractable content of C. sympodialis leaves.

Batches I II III

Moisture content (%)a 11.40 ± 0.08 (0.67%) 12.21 ± 0.10 (0.79%) 12.45 ± 0.28 (2.25%) Total ash content (%)b 9.80 ± 0.26 (2.70%) 9.10 ± 0.61 (6.68%) 9.17 ± 0.23 (2.52%) Ethanol extractable content (mg/ml)c 3.20 ± 0.40 (12.52%) 3.19 ± 0.24 (7.39%) 3.59 ± 0.18 (5.15%) Assay of warifteine (% ± SD)c 0.006 ± 0.0005 0.006 ± 0.0004 0.005 ± 0.0004 Assay of warifteine, RSD (%)c 8.33 6.66 8.80

The results were shown by average ± standard deviation (relative standard deviation); and n = 3. a Speciﬁcation: 8–14% (WHO, 1998; Brasil, 2010). b Speciﬁcation: 5–15% (WHO, 1998; Brasil, 2010). c Informative. 76 A.C. Cavalcanti et al. / South African Journal of Botany 93 (2014) 70–78

when it is necessary to apply the process of sieving to obtain plant material with a particular particle size range (Barni et al., 2009). The batches of leaves evaluated can be classiﬁed as coarse powders (Fig. 4), which are characterized, according to the Brazilian Pharmacopeia (Brasil, 2010)as having a large proportion of particles larger than 1 mm and at most 40% smaller than 0.350 mm. This information is essential for planning the extraction process using particles with standard size distribution since the yield of plant material at each particle size interval can be anticipated. The homogeneity of the particle size distribution between batches is important to conﬁrm the reproducible process of milling and the use of these leaves as raw material for obtaining a medicinal herbal product. Fig. 4. Particle size distribution of leaves of C. sympodialis.

3.6. Particle size 3.7. Warifteine content

The evaluation of the particle size of the ground material is an impor- Chemical markers are generally used at various stages of the develop- tant parameter to be set, as it exerts a direct influence on the efficiency of ment and manufacturing of a herbal medicine, such as authentication the extraction process (Migliato et al., 2007). Smaller particles increase and differentiation of species, collecting and harvesting, quality evalua- the area of contact between the solids and extractive liquid, usually mak- tion, stability assessment, diagnosis of intoxication and discovery of ing the extraction process more efficient (Sonaglio et al., 2003; Michelin lead compounds (Li et al., 2008). Ideally, chemical markers should be et al., 2010). However, the presence of very small particles can also hin- unique components that contribute to the therapeutic effects of a herbal der the processes of percolation by excessive compaction and formation medicine. As this information is scarcely available for most herbal drugs, of preferential channels (Moraes et al., 2011) or clogging of filters other chemical components are usually used as markers (Shinde et al., (Hubinger et al., 2009). The particle size distribution knowledge is useful 2009), including flavonoids, terpenoids, alkaloids and others. The amount

Fig. 5. Representative HPLC chromatograms of warifteine standard solution (10 μg/ml) (A) and of hydroalcoholic extract of C. sympodialis leaves (15 mg/ml) (B) for warifteine determination. A.C. Cavalcanti et al. / South African Journal of Botany 93 (2014) 70–78 77 of a chemical marker within a certain specified range can be an indicator Alexandre-Moreira, M.S., Piuvezam, M.R., Peçanha, L.M., 2003. Modulation of B lympho- of the quality of a herbal medicine. Chemotaxonomic markers are cyte Function by na aqueus fraction of the ethanol extract of Cissampelos sympodialis Eichl (Menispermaceae). Brazilian Journal of Medical and Biological Research 36, sometimes used as indicators of botanical identity or as tools in the 1511–1521. manufacturing of herbal medicines to help ensure the consistency of Aragão, C.F.S., Barbosa-Filho, J.M., Macedo, R.O., 2001. Thermal characterization of products. Therefore, the selection of chemical markers is crucial for the warifteine by means of TG and a DSC photovisual system. Journal of Thermal Analysis and Calorimetry 64, 185–191. quality control of herbal medicines (Ahmad et al., 2010; Braz et al., 2012). Barbosa-Filho, J.M., Agra, M.D.F., Thomas, G., 1997. Botanical, chemical and pharmacolog- Many alkaloids were isolated from C. sympodialis: warifteine (Cortes ical investigation on Cissampelos species from Paraíba (Brazil). Journal of the Brazilian et al., 1995), methylwarifteine (Barbosa-Filho et al., 1997), laurifoline, Association for the Advancement of Science 49, 386–394. Barbosa-Filho, J., Cunha, E.V.L.D., Gray, A.I., 2000. Alkaloids of the Menispermaceae. sympodialine (Alencar, 1994), milonine (Freitas et al., 1995), roraimine, Academic Press, Illinois. liriodenine (Lira et al., 2002), and desmethylroraimine (Marinho et al., Barbosa-Filho, J.M., Piuvezam, M.R., Moura, M.D., Silva, M.S., Lima, K.V.B., Cunha, E.V.L.D., 2013). The main bioactive substance present in C. sympodialis ethanol Fechine, I.M., Tekemura, O.S., 2006. Anti-inflammatory activity of alkaloids: a twenty century review. Brazilian Journal of Pharmacognosy 16, 109–139. leaf extract is warifteine, an amorphous yellow bisbenzylisoquinoline Barni, S.T., Cechinel-Filho, V., Couto, A.G., 2009. Caracterização química e tecnológica das alkaloid (Aragão et al., 2001) that occurs at a concentration of approxi- folhas, caule e planta inteira da Ipomoea pes-caprae (L.) R. Br., Convolvulaceae, como mately 1% in weight in the concentrated ethanol leaf extract (Thomas matéria-prima farmacêutica. Brazilian Journal of Pharmacognosy 19 (4), 865–870. et al., 1997b). Its pharmacological activities are similar to the action of Bezerra-Santos, C.R., Balestieri, F.M.P., Rossi-Bergmann, B., Peçanha, L.M.T., Piuvezam, M.R., 2004. Cissampelos sympodialis Eichl. (Menispermaceae): oral treatment decreases IgE aqueous fraction from the ethanolic leaf extract — AFL (Bezerra-Santos levels and induces a Th-1 shewed cytokine production in ovalbumin-sensitized et al., 2006, 2012; Costa et al., 2008; Cerqueira-Lima et al., 2010; mice. Journal of Ethnopharmacology 95, 1991–1997. Rocha et al., 2010). Since warifteine reproduces many of the pharmaco- Bezerra-Santos, C.R., Peçanha, L.M.T., Piuvezam, M.R., 2005. Cissampelos sympodialis Eichl. (Menispermaceae) inhibits anaphylactic shock reaction in murine allergic model. logical activities reported for AFL it can be considered as an important Brazilian Journal of Pharmacognosy 15, 287–291. bioactive marker for C. sympodialis AFL fraction and for plant identifica- Bezerra-Santos, C.R., Abreu, A.V.D., Barbosa-Filho, J.M., Bandeira-Mello, C., Piuvezam, M.R., tion, quality control, planning and monitoring of the technological Bozza, P.T., 2006. Anti-allergic properties of Cissampelos sympodiallis and its isolated alkaloid warifteine. International Immunopharmacology 6, 1152–1160. transformation and stability studies. Bezerra-Santos, C.R., Abreu, A.V.D., Vieira, G.C., Filho, J.R., Barbosa-Filho, J.M., Pires, A.L., The use of chromatographic and other analytical techniques that Martins, M.A., Souza, H.S., Bandeira-Mello, C., Bozza, P.T., Piuvezam, M.R., 2012. allow the separation and quantification of substances from a plant Effectiveness of Cissampelos sympodialis and its isolated alkaloid warifteine in airway hyperreactivity and lung remodeling in a mouse model of asthma. International extract is necessary to determine the chemical composition of the active Immunopharmacology 13, 148–155. raw material and also to detect potentially toxic compounds. The quan- Brasil, 2010. Farmacopéia Brasileira.V Edição. Anvisa, Brasil. tification of the chemical marker (or markers) in a plant extract is essen- Braz, R., Wolf, L.G., Lopes, G.C., Mello, J.C.P., 2012. Quality control and TLC profile data on selected plant species commonly found in the Brazilian market. Brazilian Journal of tial to allow the standardization of plant material and related products Pharmacognosy 22 (5), 1111–1118. (Drasara and Moravcova, 2004; Famei et al., 2006; Liu et al., 2007; Calixto, J.B., 2000. Efficacy, safety, quality control, marketing and regulatory guidelines for Souza-Moreira et al., 2010). Representative chromatograms of a sample herbal medicines (pythotherapeutic agents). Brazilian Journal of Medical and Biolog- – of warifteine standard and C. sympodialis ethanolic leaf extract are ical Research 33, 179 189. Capasso, R., Izzo, A.A., Pinto, L., Bifulco, T., Vitobello, C., Mascolo, N., 2000. Phytotherapy shown in Fig. 5A and B respectively. The chromatographic analysis of and quality of herbal medicines. Fitoterapia 71, S58–S65. extracts prepared from three different batches of leaves revealed similar Castro, M.M., Machado, S.R., 2006. Células e tecidos secretores. In: Appezzato-Da-Glória, B., amounts of warifteine (Table 4). Although it is difficult to establish a Carmello-Guerreiro, S.M. (Eds.), Anatomia Vegetal. UFV, Viçosa. fi Cavalcanti, A.C., Melo, I.C.A.R., Medeiros, A.F.D., Neves, M.V.M., Pereira, A.N., Oliveira, E.J., xed level for the chemical markers in a plant's extract without clinical 2013. Studies with Cissampelos sympodialis: the search towards the scientificvalida- data, the preparation and use of standardized extracts during clinical tri- tion of a traditional Brazilian medicine used for the treatment of asthma. Brazilian als is essential and our work can provide the tools to the preparation of Journal of Pharmacognosy 23 (3), 527–541. Cerqueira-Lima, A.T., Alcântara-Neves, N.M., Carvalho, L.C.P.D., Barbos-Filho, J.M., C. sympodialis extracts with known warifteine content. Piuvezam, M.R., Russo, M., Barboza, R., Oliveira, E.D., Marinho, A., Figueiredo, C.A., 2010. Effects of Cissampelos sympodialis Eichl. and its alkaloid, warifteine, in an exper- imental model of respiratory allergy to Blomia tropicalis. Current Drug Targets 11, 4. Conclusion 1458–1467. Correa, M.P., 1984. Dicionário de plantas úteis do Brasil e das exóticas cultivadas. Given the preclinical results obtained with the aqueous fraction Ministério da Agricultura, IBDF, Brasil. Corrêa, M.F.P., Melo, G.O.D., Costa, S.S., 2008. Substâncias de origem vegetal of the hydroalcoholic extract from the leaves of C. sympodialis and the potencialmente úteis na terapia da asma. Brazilian Journal of Pharmacognosy 18, lack of data regarding the quality control of its leaves, the results 785–797. presented here will be important to ensure that the plant material Cortes,S.F.,Alencar,J.L.D.,Thomas,G.,Barbosa-Filho,J.M.,1995.Spasmolytic actions of warifteine, a bisbenzylisoquinoline alkaloid isolated from the root bark of meet reproducible quality standards. Thus, it is possible to suggest a Cissampelos sympodialis Eichl (Menispermaceae). Phytotherapy Research 9, monograph for the quality control of C. sympodialis leaves. This is a 579–583. fundamental necessity if further investigations with clinical trials are Costa, H.F., Bezerra-Santos, C.R., Barbosa-Filho, J.M., Martins, M.A., Piuvezam, M.R., 2008. Warifteine, a bisbenzylisoquinoline alkaloid, decreases immediate allergic and thermal to be conducted aiming at developing a herbal medicine for the treat- hyperalgesic reactions in sensitized animals. International Immunopharmacology 8, ment of asthma. 519–525. 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