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Microscopic and UV/Vis spectrophotometric characterization of Cissampelos pareira of Brazil and Africa

ABSTRACT

Cissampelos pareira L., belonging to Menispermaceae family, has worldwide distribution, occurring in tropical and subtropical regions of the Americas, Africa and Asia. It is the most popular species of Cissampelos, known for its medicinal uses of leaves and roots. The study aims to find distinctive leaf anatomical characters, and also demonstrate the importance of spectral data to identify C. pareira samples, in order to contribute to its taxonomy and quality control of its drugs. Anatomical leaf analyses were performed by optical and scanning electron microscopy. The spectral profile was obtained from methanolic extracts of C. pareira samples from Brazil and Africa, with application of UV–vis spectrophotometry data, which were analyzed by principal component analysis (PCA). Some anatomical characters such as leaf epidermal cells walls, stomata, trichomes, mesophyll, features of midrib and petiole, and the spectral profile within the wavelength ranging between 770 and 240 nm (eight bands) differs between Brazilian and African samples. The results represent an additional support to the taxonomy of C. pareira, and the quality control of their leaf drugs, mainly in relation to misidentified samples.

Keywords:
Ethnomedicine; Leaf anatomy; Medicinal plant; Menispermaceae; Morphoanatomy; Pharmacobotany

Introduction

Cissampelos pareira L., belonging to Menispermaceae family, has worldwide distribution, occurring in tropical and subtropical regions of the Americas, Africa and Asia (Ortiz, 2001Ortiz, R., 2001. Menispermaceae. In Stevens, W.D., Ulloa Ulloa, C., Pool, A., Montiel, O. M. (orgs.), Flora de Nicaragua. Missouri: Monographs in Systematic Botany from the Missouri Botanical Garden, p. 1432–1442.). In Brazil, it is encountered in different types of vegetation, from Caatinga, Atlantic Forest and Amazon forest (Braga, 2015Braga, J.M.A. Menispermaceae in Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro. http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB10031 (accessed July 2015).
http://floradobrasil.jbrj.gov.br/jabot/f...
). According to Schmelzer and Gurib-Fakim (2008)Schmelzer, G.H., Gurib-Fakim, A. (Eds.), 2008. Plant Resources of Tropical Africa 11(1). Medicinal Plants 1. PROTA Foundation/Backhuys Publishers/CTA, Wageningen, Netherlands/Leiden, Netherlands/Wageningen, Netherlands, 791 pp., in Africa this species occurs in subtropical forest, savannah, deciduous shrubs, often persisting in cleared land and plantations, also in secondary vegetation and near rock outcrops.

It is the most popular species of Cissampelos not only for its wide distribution, but mainly because its leaves and roots are widely used as medicinal. According to Napralert (2013)NAPRALERT, Natural Products Alert. http://www.napralert.org/ (accessed March 2013).
http://www.napralert.org/...
, C. pareira has more than eighty folk names. In Brazil, it is known as "parreira", "abuta", and "parreira-brava" (Lewis and Elvin-Lewis, 1977Lewis, W.H., Elvin-Lewis, M.P.F., 1977. Medical Botany. Wiley-Interscience, New York.; Rury, 1983Rury, P.M., 1983. Pareira Brava: 19th Century notes and commercial samples from E. R. Squibb M. D.. Bot. Mus. Leaf. 29, 27-48.); in Africa, it is called in folk medicine as "chegonde" and "karigi-munana" (Hedberg et al., 1983Hedberg, I., Hedbrerg, O., Madati, P.J., Mshigeni, K.E., Mshiu, E.N., Samuelsson, G., 1983. Inventory of plants used in traditional medicine in Tanzania. II. Plants of the families Dilleniaceae-Opiliaceae. J. Ethnopharmacol. 9, 105-127.; Rukunga et al., 2009Rukunga, G.M., Gathirwa, J.W., Omar, S.A., Muregi, F.W., Muthaura, C.N., Kirira, P.G., Mungai, G.M., Kofi-Tsekpo, W.M., 2009. Anti-plasmodial activity of the extracts of some Kenyan medicinal plants. J. Ethnopharmacol. 121, 282-285.); and in India, it is known as "ambastha", "patha" and "laghupatha" (Vaidya, 1988Vaidya, B.G., 1988. Nighantu Adarsh, vol. 1. Chaukhambha Bharti Academy Publications, Varanasi, India, pp. 44–45.).

In many ethnobotanical reports, the leaves of C. pareira are recognized as a natural medicine for various purposes. The leaf juice is used as antiseptic, anthelmintic, insecticidal and parasiticidal, and against dermatitis (Singh and Ali, 1992Singh, V.K., Ali, Z.A., 1992. A contribution to the ethnopharmacological study of the Udaipur forests of Rajasthan, India. Fitoterapia 63, 136-144.), asthmas (Singh and Maheshwari, 1994Singh, K.K., Maheshwari, J.K., 1994. Traditional phytotherapy of some plants used by the tharus of the Nainital District, Uttar Pradesh, India. Int. J. Pharmacogn. 32, 51-58.), genitourinary disorders (Sanchez Medina et al., 2001Sanchez Medina, A., Garcia Sosa, K., May Pat, F., Pena Rodriguez, L.M., 2001. Antioxidant, antimicrobial and beta-glucosidase inhibition activities. Phytomedicine 8, 144-151.), diarrhea, dysenteries and gastrointestinal disorders (Kumar et al., 2006Kumar, V.P., Chauhan, N.S., Padh, H., Rajani, M., 2006. Search for antibacterial and antifungal agents from selected Indian medicinal plants. J. Ethnopharmacol. 107, 182-188.; Kamble et al., 2008Kamble, S.Y., More, T.N., Patil, S.R., Pawar, S.G., Ram Bindurani Bodhankar, S.L., 2008. Plants used by tribes of Northwest Maharashtra for the treatment of gastrointestinal disorders. Indian J. Tradit. Knowl. 7, 321-325.), antifertility (Ganguly et al., 2007Ganguly, M., KrBorthakur, M., Devi, N., Mahanta, R., 2007. Antifertility activity of the methanolic leaf extract of Cissampelos pareira in female albino mice. J. Ethnopharmacol. 111, 688-691.; Priya et al., 2012Priya, G., Saravanan, K., Renuka, C., 2012. Medicinal plants with potential antifertility activity – a review of sixteen years of herbal medicine research (1994–2010). Int. J. Pharm. Tech. Res. 4, 481-494.), and antidiabetic (Yadav et al., 2013Yadav, K.S., Yadav, N.P., Shanker, K., Thomas, S.C., Srivastav, S., Srivastava, S., Kumar Rai, V., Mishra, N., Sinha, P., 2013. Assessment of antidiabetic potential of Cissampelos pareira leaf extract in streptozotocinenicotinamide induced diabetic mice. J. Pharm. Res. 6, 874-878.). The topical use of leaves is indicated to treat hemorrhages from cuts, burns and wounds (Ramasubramaniaraja and Babu, 2010Ramasubramaniaraja, R., Babu, N.M., 2010. Antihelminthic studies and medicinal herbs – an overview. Int. J. Pharm. Sci. Rev. Res. 5, 39-47.; Shukla et al., 2012Shukla, P., Shukla, P., Gopalakrishna, B., 2012. Investigation of in-vitro anthelmintic activity of Cissampelos pareira Linn against Pheretima posthuma. Int. J. Pharm. Sci. Res. 3, 265-267.), and also to treat abscesses (Abbasi et al., 2010Abbasi, A.M., Khan, M.A., Ahmad, M., Zafar, M., Jahan, S., Sultana, S., 2010. Ethnopharmacological application of medicinal plants to cure skin diseases and in folk cosmetics among the tribal communities of North-West Frontier Province, Pakistan. J. Ethnopharmacol. 128, 322-335.; Haque et al., 2011Haque, M.A., Shaha, M.K., Ahmed, S.U., Akter, R., Rahman, H., Chakravotry, S., Imran, A.H.M.N., Islam, M.T., Das, R.C., Rahmatullah, M., 2011. Use of inorganic substances in folk medicinal formulations: a case study of a folk medicinal practitioner in Tangail district, Bangladesh. Am. Eurasian J. Sustain. Agric. 5, 415-423.). In addition, in India, the leaves are also used as cattle feed to increase milk production, and also in some food systems as thickeners, gelling agents, texture modifiers and stabilizers (Vardhanabhuti and Ikeda, 2006Vardhanabhuti, B., Ikeda, S., 2006. Isolation and characterization of hydrocolloids from monoi (Cissampelos pareira) leaves. Food Hydrocolloids 20, 885-891.; Priya et al., 2012Priya, G., Saravanan, K., Renuka, C., 2012. Medicinal plants with potential antifertility activity – a review of sixteen years of herbal medicine research (1994–2010). Int. J. Pharm. Tech. Res. 4, 481-494.), inter alia.

The leaves of C. pareira have been reported to be a rich source of isoquinoline and bisbenzylisoquinoline alkaloids (Shukla et al., 2012Shukla, P., Shukla, P., Gopalakrishna, B., 2012. Investigation of in-vitro anthelmintic activity of Cissampelos pareira Linn against Pheretima posthuma. Int. J. Pharm. Sci. Res. 3, 265-267.), such as berberine (Kupchan et al., 1960aKupchan, S.M., Yokoyama, N., Beal, J.L., 1960a. Menispermaceae alkaloids. I. The alkaloids of Cissampelos pareira Linn. and the origin of radix pareira brave. J. Am. Pharm. Assoc. 49, 727.), curine (Chowdhury, 1972Chowdhury, A.R., 1972. Chemical investigations on Cissampelos pareira. Sci. Cult. 38, 358.), hayatine (Sharma, 1987Sharma, V., 1987. Biosynthesis of hayatin. Indian J. Chem. 26, 589-591.) and magnoflorine (Ahmad et al., 1992Ahmad, R., Malik, M.A., Zia-ul-Haq, M., 1992. Alkaloids of Cissampelos pareira. Fitoterapia 63, 282-285.). In addition, have also been isolated essential oil (Kupchan et al., 1960bKupchan, S.M., Slade, P., Young, R.J., 1960b. Intramolecular catalysis. Facilitation of alkaline hydrolysis of alicyclic 1,2-diol monoesters. Tetrahedron Lett. 1, 22-25.), flavonoids (Ramirez et al., 2003Ramirez, I., Carabot, A., Melendez, P., Carmona, J., Jimenez, M., Patel, A.V., Crab, T.A., Blunden, G., Cary, P.D., Croft, S.L., Costa, M., 2003. Cissampeloflavone, a chalcone–flavone dimer from Cissampelos pareira . Phytochemistry 64, 645-647.; Amresh et al., 2007aAmresh, G., Zeashan, H., Rao, C.V., Singh, P.N., 2007a. Prostaglandin mediated anti-inflammatory and analgesic activity of Cissampelos pareira. Acta Pharm. Sci. 49, 153-160.), polysaccharides (Vardhanabhuti and Ikeda, 2006Vardhanabhuti, B., Ikeda, S., 2006. Isolation and characterization of hydrocolloids from monoi (Cissampelos pareira) leaves. Food Hydrocolloids 20, 885-891.), and pectin (Singthong et al., 2004Singthong, J., Cui, S.W., Ningsanond, S., Douglas Goff, H., 2004. Structural characterization, degree of esterification and some gelling properties of Krueo Ma Noy (Cissampelos pareira) pectin. Carbohydr. Polym. 58, 391-400.; Arkarapanthu et al., 2005Arkarapanthu, A., Chavasit, V., Sungpuag, P., Phuphathanaphong, L., 2005. Gel extracted from Khruea-ma-noi (Cyclea barbata Miers) leaves: chemical composition and gelation properties. J. Sci. Food Agric. 85, 1741-1749.) have also been isolated.

Biological and pharmacological activities of leaves and aerial parts (leaves and branches) of C. pareira were demonstrated in several studies. The cissampeloflavone, isolated from leaves, showed activity against Trypanosoma cruzi and T. brucei rhodesiense (Ramirez et al., 2003Ramirez, I., Carabot, A., Melendez, P., Carmona, J., Jimenez, M., Patel, A.V., Crab, T.A., Blunden, G., Cary, P.D., Croft, S.L., Costa, M., 2003. Cissampeloflavone, a chalcone–flavone dimer from Cissampelos pareira . Phytochemistry 64, 645-647.). The plant extract exhibited antifungal activity against Aspergillus niger and Saccharomyces cerevisiae (Kumar et al., 2006Kumar, V.P., Chauhan, N.S., Padh, H., Rajani, M., 2006. Search for antibacterial and antifungal agents from selected Indian medicinal plants. J. Ethnopharmacol. 107, 182-188.). The ethanol extract of the aerial parts showed anti-inflammatory and analgesic activities (Amresh et al., 2007bAmresh, G., Zeashan, H., Gupta, R.J., Kant, R., Rao, C.V., Singh, P.N., 2007b. Gastroprotective effects of ethanolic extract from Cissampelos pareira in experimental animals. J. Nat. Med. 61, 323-328.). The contraceptive and cytotoxic effects were demonstrated by Priya et al. (2012)Priya, G., Saravanan, K., Renuka, C., 2012. Medicinal plants with potential antifertility activity – a review of sixteen years of herbal medicine research (1994–2010). Int. J. Pharm. Tech. Res. 4, 481-494. and Ganguly et al. (2007)Ganguly, M., KrBorthakur, M., Devi, N., Mahanta, R., 2007. Antifertility activity of the methanolic leaf extract of Cissampelos pareira in female albino mice. J. Ethnopharmacol. 111, 688-691., respectively. The anti-diabetic activity was confirmed by Jannu et al. (2011)Jannu, V., Sai Vishal, D., Ranjith Babu, V., Harisha, B., Ravi Chandra Sekhara Reddy, D., 2011. Antidiabetic activity of hydro-alcoholic extract of Cissampelos pareira Linn. Leaves in streptozotocin induced diabetic rats. Int. J. Pharm. Technol. 3, 3601-3611. and Yadav et al. (2013)Yadav, K.S., Yadav, N.P., Shanker, K., Thomas, S.C., Srivastav, S., Srivastava, S., Kumar Rai, V., Mishra, N., Sinha, P., 2013. Assessment of antidiabetic potential of Cissampelos pareira leaf extract in streptozotocinenicotinamide induced diabetic mice. J. Pharm. Res. 6, 874-878.. In addition, a preliminary study carried out by Thakur and Rana (2013)Thakur, P., Rana, A.C., 2013. Effect of Cissampelos pareira leaves on anxiety-like Behavior in experimental animals. J. Tradit. Complement. Med. 3, 188-193. confirmed the anxiolytic effect of C. pareira leaves.

According to Rhodes (1975)Rhodes, D.G., 1975. A revision of the genus Cissampelos. Phytologia 30, 415-485. and Hoot et al. (2009)Hoot, S.B., Zautke, H., Harris, D.J., Crane, P.R., Neves, S.S., 2009. Phylogenetic patterns in Menispermaceae based on multiple chloroplast sequence data. Syst. Bot. 34, 44-56., C. pareira has problems in its interspecific delimitation with imprecise limits, mainly caused by its wide distribution and great plasticity of their vegetative forms. On the other hand, the leaf anatomical studies have shown to be an additionall support to the plant taxonomy, as already done in Solanum (Nurit-Silva et al., 2007Nurit-Silva, K., Basílio, I.J.L.D., Agra, M.F., 2007. Estudo farmacobotânico comparativo entre Solanum paniculatum L. e Solanum rhytidoandrum Sendtn. Rev. Bras. Biol. 5, 243-245.; Nurit-Silva and Agra, 2011Nurit-Silva, K., Agra, M.F., 2011. Leaf epidermal characters of Solanum sect. polytrichum (Solanaceae) as taxonomic evidence. Microsc. Res. Tech. 74, 1186-1191.; Sampaio et al., 2014Sampaio, V.S., Araújo, N.D., Agra, M.F., 2014. Characters of leaf epidermis of Solanum of the Brevantherum Clade from Atlantic Forest of Northeastern Brazil. S. Afr. J. Bot. 74, 108-113.), and also to the Menispermaceae family, including Cissampelos by De Wet et al. (2002)De Wet, H., Tilney, P.M., Van Wyk, B.E., 2002. Vegetative morphology and anatomy of Cissampelos in South Africa. S. Afr. J. Bot. 68, 181-190., Porto et al. (2008Porto, N.M., Basílio, I.J.L.D., Agra, M.F., 2008. Pharmacobotanical study of the leaves of Cissampelos sympodialis Eichl., (Menispermaceae). Braz. J. Pharmacogn. 18, 102-107., 2011Porto, N.M., Figueiredo, R.C.B.Q., Oliveira, A.F.M., Agra, M.F., 2011. Leaf epidermal characteristics of Cissampelos L. (Menispermaceae) species from Northeastern Brazil. Microsc. Res. Tech. 74, 370-376., 2012)Porto, N.M., Araújo, N.D., Basílio, I.J.L.D., Agra, M.F., 2012. Analysis of leaf epidermal characters of medicinal and poisonous Brazilian Menispermaceae. Planta Med. 78, 1111., for example.

The spectroscopic chemical techniques have emerged and contributed as an additional tool to contribute to plant taxonomy, and also as a support to the quality control of herbal drugs, allowing information to be obtained without the need for previous isolation of chemical constituents, as demonstrated before for Baccharis (Lonni et al., 2005Lonni, A.A.S.G., Scarminio, I.S., Silva, L.M.C., Ferreira, D.T., 2005. Numerical taxonomic characterization of Baccharis genus species by ultravioleta-visible spectrophotometry. Anal. Sci. 21, 235-239.) and Solanum (Basílio et al., 2012Basílio, I.J.L.D., Bhattacharyya, J., Moura, R.K.P., Agra, M.F., 2012. Application of UV/VIS spectrophotometry and multivariate analysis to characterization of the species of Solanum sect. Erythrotrichum CHILD. Chem. Biodivers. 9, 1114-1124.).

Although the leaves of C. pareira are commonly used in traditional medicine, and there is evidence of many activities of their compounds, a literature survey showed a lack of studies of the leaf comparative anatomy, as well as spectroscopic analysis of UV–visible of the leaf extracts. In this way, this study aimed to find leaf anatomical characters, distinctive to C. pareira, on samples of plants from Brazil and Africa, revealing the importance of anatomical studies combined with spectral data, would be useful to the quality control of its drugs, as well as to the taxonomy of C. pareira.

Materials and methods

Plant material

Botanical expeditions and field observations were carried out by N.M. Porto, in areas of Atlantic Forest and Rain Forest, for sample collection of Menispermaceae, including leaves of Cissampelos pareira L. in the following Brazilian States: Alagoas, Pará Maranhão, Paraíba, Pernambuco and Sergipe (Table 1). For each individual, an average of three leaf samples were taken from the second to the fifth nodes of the leaf blades and the proximal, median and distal portions, and petiole were fixed in FAA (50%) for 24 h (Johansen, 1940Johansen, D.A., 1940. Plant Microtechnique. McGraw Hill, New York.), and preserved in ethanol 70 GL. The other part of fertile material was pressed and dried for herbaria, according to Bridson and Forman (1999)Bridson, D., Forman, L., 1999. The Herbarium Handbook, 3rd ed. Royal Botanic Gardens, Kew.. The voucher specimens were deposited at the Herbarium Prof. Lauro Pires Xavier (JPB), of the Universidade Federal da Paraíba.

Table 1
Selected voucher specimens of Cissampelos pareira and species of outgroup.

In addition, leaf samples from herbarium specimens identified as C. pareira were also analyzed from the following herbaria, acronyms by Thiers (2015)Thiers, B. (continuously updated) Index Herbariorum: A Global Directory of Public Herbaria and Associated Staff. New York Botanical Garden's Virtual Herbarium. New York Botanical Garden. Available from: http://sweetgum.nybg.org/ih/ (accessed 29.10.15).
http://sweetgum.nybg.org/ih/...
: Herbarium of Centro de Pesquisas do Cacau (CEPEC), Herbarium Prof. Jayme Coelho de Morais (EAN), Herbarium of Embrapa Amazônia Oriental (IAN), Herbário Prof. Lauro Pires Xavier (JPB), Herbário Museu Paraense Emílio Goeldi (MG), Herbarium Jardim Botânico do Rio de Janeiro (RB), Herbarium Prof. Geraldo Mariz (UFP) of the Universidade Federal de Pernambuco (UFPE), and Herbarium of Missouri Botanical Garden (MO). A list of voucher specimens used in this study is given in Table 1.

Anatomical and histochemical analysis

The plant material was divided in two portions, one for analysis by optical microscopy and the other by scanning electron microscopy (SEM). Transverse sections were performed on leaves of C. pareira by free hand using commercial razor blades. Subsequently, the sections were cleared by sodium hypochlorite (20%) until complete clarification, neutralized with acetic acid (0.2%), washed in distilled water, and stained with a solution of Astra blue and Safranin, modified by Bukatsch (1972)Bukatsch, F., 1972. Azul de Astra e Safranina. In: Kraus, J., Arduin, M. (Eds.), Manual básico de métodos em morfologia vegetal. Edur, Seropédia, Rio de Janeiro, p. 26.. Leaf epidermis was separated from the mesophyll by dissociation in Jeffrey solution (Johansen, 1940Johansen, D.A., 1940. Plant Microtechnique. McGraw Hill, New York.), and then stained with Safranin with 1% solution in 50% alcohol, according to Franklin (1945)Franklin, G.L., 1945. Preparation of thin sections of synthetic resins and wood-resin composites, and a new macerating method for wood. Nature 155, 51..

For alkaloid detection, transverse sections were treated with Dittman's and Wagner reagents (Furr and Mahlberg, 1981Furr, M., Mahlberg, P.G., 1981. Histochemical analyses of laticifers and glandular trichomes in Cannabis sativa. J. Nat. Prod. 44, 153-159.). All leaf sections were mounted in glycerinated gelatin (50%). The observations and microphotographs were performed by a photomicroscope (Leica DM750), with image processing software (Qwin System) coupled to a video camera (Leica ICC50 HD) for image capture.

Scanning electron microscopy (SEM)

Scanning electron microscopy (SEM) of leaf epidermis was performed in dry material to optimize the observation of waxes and epidermal appendages. Leaf fragments of 1 cm2 were fixed in a solution of 4% glutaraldehyde in 0.1 M potassium phosphate buffer (pH 7.0) for 24 h, at 48 ºC, then washed in 0.1 M sodium cacodylate buffer (pH 7.0), followed by post fixation in 1% OsO4, in 0.1 M Na-cacodylate buffer (pH 7.0) for 1 h, at room temperature. Subsequently, the fragments were dehydrated in a crescent ethylic series, and dried at the critical point, placed on aluminum stubs with double-sided tape, air-dried and, finally, coated with gold. Finally, photomicrographs and microscopic analysis were performed by scanning electron microscopy (JEOL JSM-5600), on leaf epidermis, at an accelerating voltage of 15 KV. Micromorphological characterization was complemented by the analysis of epicuticular waxes of the leaf epidermis that were classified here according to Barthlott et al. (1998)Barthlott, W., Neinhuis, C., Cutler, D., Ditsch, F., Meusel, I., Theisen, I., Wilhelmi, H., 1998. Classification and terminology of plant epicuticular waxes. J. Linn. Soc. 126, 237-260..

Chemical analysis

Samples of dried leaves of C. pareira (from Brazil and Africa) and of nine species used as an outgroup of Menispermaceae family were investigated: Cissampelos andromorpha DC., Cissampelos sympodialis Eichl., Cissampelos tropaeolifolia DC., Anomospermum chloranthum Diels, Anomospermum steyermarkii Krukoff & Barneby, Hyperbaena domingensis (DC.) Benth., Orthomene hirsuta (Krukoff & Moldenke) Barneby & Krukoff, Orthomene schomburgkii (Miers) Barneby & Krukoff, Sciadotenia brachypoda Diels. All UV spectrophotometric analysis were performed using a modified version of a previously published method (Basílio et al., 2012Basílio, I.J.L.D., Bhattacharyya, J., Moura, R.K.P., Agra, M.F., 2012. Application of UV/VIS spectrophotometry and multivariate analysis to characterization of the species of Solanum sect. Erythrotrichum CHILD. Chem. Biodivers. 9, 1114-1124.). Briefly, methanol extracts of dried leaves were prepared by ultrasound extraction for 20 min at room temperature, and then filtered through membranes with a pore size of 0.45 mm, modified from Basílio et al. (2012)Basílio, I.J.L.D., Bhattacharyya, J., Moura, R.K.P., Agra, M.F., 2012. Application of UV/VIS spectrophotometry and multivariate analysis to characterization of the species of Solanum sect. Erythrotrichum CHILD. Chem. Biodivers. 9, 1114-1124..

The UV/Vis spectra were recorded with a spectrophotometer (UV-1650PC, Shimadzu, Kyoto, Japan). For all absorbance measurements Quartz cells (1 cm) were used. The spectra were recorded in triplicate from 770 to 200 nm. The standardized procedure was repeated for all species and the data was automatically reduced to an ASCII file.

The spectra were normalized by setting the absorbance at 770 nm equal to zero and subsequently mean centered. The data matrix was processed by FITOPAC v.2.1.2 software. Finally, a principal component analysis (PCA) was performed using the methods variance-covariance between the groups with Past software, version 2.15 (Hammer et al., 2001Hammer, Ø., Harper, D.A.T., Ryan, P.D., 2001. PAST: Paleontological statistics software package for education and data analysis. Palaeontol. Electron. 4, 1-9.).

Results and discussion

Anatomic study

The leaf epidermis of C. pareira, in front view, presented cells with curve to waved anticlinal walls on the glabrescent adaxial surface (Fig. 1A and C), and waved and hairy on the abaxial surface (Table 2, Fig. 1B and D). In transverse section, the epidermis of C. pareira was uniseriate with tabular cells, and a thin and smooth cuticle (Fig. 3A). Metcalfe and Chalk (1950)Metcalfe, C.R., Chalk, L., 1950. Anatomy of Dicotyledons: Leaves, Stem, and Woods in Relation to Taxonomy with Notes on Economic Uses, vol. 1. Clarendon Press, Oxford, United Kingdom, pp. 52–62., Porto et al. (2011)Porto, N.M., Figueiredo, R.C.B.Q., Oliveira, A.F.M., Agra, M.F., 2011. Leaf epidermal characteristics of Cissampelos L. (Menispermaceae) species from Northeastern Brazil. Microsc. Res. Tech. 74, 370-376. and Sudhakaran (2012)Sudhakaran, M.V., 2012. Histo-morphological, fluorescent and powder microscopic characterization of Cissampelos pareira Linn. Phcog. J. 4, 57-68. have reported this pattern of cell walls to C. pareira. It was also recorded in other species of Cissampelos (Hong et al., 2001Hong, Y.P., Pan, K.Y., Chen, Z.D., Lu, A.M., 2001. Characters of leaf epidermis and their systematic significance in Menispermaceae. Acta Bot. Sin. 43, 615-623.; De Wet et al., 2002De Wet, H., Tilney, P.M., Van Wyk, B.E., 2002. Vegetative morphology and anatomy of Cissampelos in South Africa. S. Afr. J. Bot. 68, 181-190.; Porto et al., 2011Porto, N.M., Figueiredo, R.C.B.Q., Oliveira, A.F.M., Agra, M.F., 2011. Leaf epidermal characteristics of Cissampelos L. (Menispermaceae) species from Northeastern Brazil. Microsc. Res. Tech. 74, 370-376.), and other genera of Menispermaceae as Cocculus and Stephania (Metcalfe and Chalk, 1950Metcalfe, C.R., Chalk, L., 1950. Anatomy of Dicotyledons: Leaves, Stem, and Woods in Relation to Taxonomy with Notes on Economic Uses, vol. 1. Clarendon Press, Oxford, United Kingdom, pp. 52–62.). Only two samples from Brazil (CP6 and CPb), and two from Africa (CP8 and CP9) showed thickened cuticle (Figs. 4E and 5C, E) that differed from the C. pareira pattern (Table 2). To Wilkinson (1979)Wilkinson, H.P., 1979. Plant surface. In: Metcalfe, C.R., Chalk, L. (Eds.), Anatomy ofthe Dicotyledon. Claredon Press, Oxford, pp. 97–162., in general the presence and thickness of the cuticle is determined by environmental factor and does not have taxonomic importance.

Fig. 1
Cissampelos pareira (MF Agra 5061), front view: (A) adaxial surface laxe-pilose; (B) abaxial surface pubescent; (C) leaf epidermis with waved anticlinal walls cells on the adaxial surface by light microscopy; (D) Leaf epidermis with stomata (st1, anomocytic and st2, anisocytic) on the abaxial surface by light microscopy.
Fig. 2
Cissampelos pareira (RL Fróes 30443), leaf epidermis in front view by SEM: (A) adaxial surface with long finger hairs; (B) detail of the wrinkled epidermis and trichomes on the abaxial surface; (C) detail of the abaxial epidermis with epicuticular waxes, as tubule; (D) stoma in detail and epicuticular waxes on the abaxial surface (Legends: epw, epicuticular wax; st, stomata; tri, trichome).
Fig. 3
Cissampelos pareira (AL Gasper 2020) in transverse sections: (A) dorsiventral mesophyll; (B) midrib with collateral vascular bundle; (C) vascular bundles of petiole in the basal portion; (D) vascular bundles of petiole in the median portion with a sclerenchymatous ring; (E) detail of collateral vascular bundle; (F) stained idioblasts showing positive reaction for alkaloids (Legends: col, collenchyma; ef, exchange fascicular; ep, epidermis; fc, fascicular cambium; fp, fundamental parenchyma; id, idioblast; per, pericycle; ph, phloem; pp, palisade parenchyma; pt, pith; rsc, sclerenchymatous ring; scl, sclerenchyma; sp, spongy parenchyma; st, stomata; tri, trichome; vb, vascular bundle; xyl, xylem).
Fig. 4
(A, B) Cissampelos sp1 (HS Irwin s/n – IAN), leaf epidermis in front view: (A) Leaf epidermis with straight anticlinal cells walls on the adaxial surface; (B) Leaf epidermis with curve anticlinal cells walls on the abaxial surface; (C–H) Cissampelos sp2 (AC Cervi 3276), transverse sections: (C) leaf margin somewat rounded; (D) Plane convex midrib with collateral vascular bundle; (E) Petiole of 12 collateral vascular bundles in the basal portion; (F) detail of parenchyma with styloids cristals, (G) detail of epidermal papillae on the abaxial surface; (H) detail of mesophyll with secretory cavities; (Legends: cr, crystals; ep, epidermis; pap, papillae; pp, palisade parenchyma; pt, pith; sc, secretory cavity; scl, sclerenchyma; sct, scar of trichome; sp, spongy parenchyma; st, stomata; tri, trichome; vb, vascular bundle).
Fig. 5
Cissampelos sp3, transverse sections of leaf blades. (A) (JW Ash 655): detail of epidermal papillae; (B–D) (PK Rwaburindore 205): (B) detail of a stoma on the abaxial surface; (C) dorsiventral mesophyll with bisseriate palisade; (D) plane-convex midrib with collateral vascular bundle; (E) (H Faulkner 5631): detail of mesophyll with secretory cavities; (F, H) (PK Rwaburindore 205): (F) Palisade with secretory cavities; (G, H) stained secretory cavities showing positive reaction for alkaloids (Legends: ct, cuticle; ep, epidermis; pap: papillae; pp, palisade parenchyma; sc, secretory cavity; sp, spongy parenchyma; st, stomata; tri, trichome; vb, vascular bundle).

Table 2
Selected leaf anatomical characters of Cissampelos pareira L. and related, and their states.

Eight specimens identified as C. pareira displayed the leaf epidermis pattern different from those known for this species. Two samples from Brazil (CP6 and CPb) showed straight anticlinal walls on the adaxial, and curve on the abaxial (Table 2, Fig. 4C and D). According to Stace (1965)Stace, C.A., 1965. Cuticular studies as an aid to plant taxonomy. Bull. Br. Mus. Nat. Hist. Bot. 4, 1-78., the features of anticlinal walls are a mesomorphic character, and environmental conditions such as humidity play a significant role in determining the pattern of anticlinal cell walls. These features also have been reported to vary in response to changes in light regimes by Rôças et al. (2001)Rôças, G., Scarano, F.R., Barros, C.F., 2001. Leaf anatomical variation in Alchornea triplinervia (Spreng.) Müll. Arg. (Euphorbiaceae) under distinct light and soil water regimes. Bot. J. Linn. Soc. 136, 1-8. and Mantuano et al. (2006)Mantuano, D.G., Barros, C.F., Scarano, F.R., 2006. Leaf anatomy variation within and between three restinga populations of Erythroxylum ovalifolium Peyr. (Erythroxylaceae) in Southeast Brazil. Rev. Bras. Bot. 29, 209-215..

Four samples from Brazil, CP3, CP5, CPa and CPc (Table. 2, Fig. 4G), and three from Africa (CP7, CP8 and CP9) showed an epidermal papillae (Table. 2, Fig. 5A). The presence, number and distribution of papillae constitute an important feature that has been used to define boundaries at specific and generic level in many taxonomic groups (Hong et al., 2001Hong, Y.P., Pan, K.Y., Chen, Z.D., Lu, A.M., 2001. Characters of leaf epidermis and their systematic significance in Menispermaceae. Acta Bot. Sin. 43, 615-623.). According to Bone et al. (1985)Bone, R.E., Lee, D.W., Norman, J.M., 1985. Epidermal cells functioning as lenses in leaves of tropical rainforest shade plants. Appl. Opt. 24, 1408-1412., the cell walls of the leaf epidermis with convex curvature would be advantageous to increase the energy capture efficiency, which is important for plants that must survive at extremely low light levels. Furthermore, papillae in the leaf epidermis minimize the area of contact causing a very low adhesion of water on leaf (Ensikat et al., 2011Ensikat, H.J., Ditsche-Kuru, P., Neinhuis, C., Barthlott, W., 2011. Superhydrophobicity in perfection: the outstanding properties of the lotus leaf. Beilstein J. Nanotechnol. 2, 152-161.).

Epicuticular waxes as tubules and clusters of tubules were confirmed in SEM, in both surfaces of C. pareira (Fig. 2C and D), as previously referred by Porto et al. (2011)Porto, N.M., Figueiredo, R.C.B.Q., Oliveira, A.F.M., Agra, M.F., 2011. Leaf epidermal characteristics of Cissampelos L. (Menispermaceae) species from Northeastern Brazil. Microsc. Res. Tech. 74, 370-376.. The epicuticular waxes also have taxonomic value in the characterization of the leaf epidermis, according to Barthlott et al. (1998)Barthlott, W., Neinhuis, C., Cutler, D., Ditsch, F., Meusel, I., Theisen, I., Wilhelmi, H., 1998. Classification and terminology of plant epicuticular waxes. J. Linn. Soc. 126, 237-260.. Moreover, the presence of wax tubules in leaf epidermis could be related to the higher water repellency (Ensikat et al., 2011Ensikat, H.J., Ditsche-Kuru, P., Neinhuis, C., Barthlott, W., 2011. Superhydrophobicity in perfection: the outstanding properties of the lotus leaf. Beilstein J. Nanotechnol. 2, 152-161.).

The leaves were hypostomatic with anomocytic and anisocytic stomata occurring simultaneously, both at the epidermal level (Fig. 3A), however, the anomocytic type was predominant (Fig. 1D), which corroborate with the pattern previously described to C. pareira by De Wet et al. (2002)De Wet, H., Tilney, P.M., Van Wyk, B.E., 2002. Vegetative morphology and anatomy of Cissampelos in South Africa. S. Afr. J. Bot. 68, 181-190. and Porto et al. (2011)Porto, N.M., Figueiredo, R.C.B.Q., Oliveira, A.F.M., Agra, M.F., 2011. Leaf epidermal characteristics of Cissampelos L. (Menispermaceae) species from Northeastern Brazil. Microsc. Res. Tech. 74, 370-376.. Simple bi-celled to many-celled trichomes were present on both surfaces of the leaf epidermis (Figs. 1A, B and 2A, B), which are common on the leaf epidermis of some Menispermaceae species, according to Wilkinson (1978)Wilkinson, P., 1978. Leaf anatomy of the Tribe Coscinieae Hook. f. & Thoms. (Menispermaceae). Kew Bull. 32, 347-360..

Dorsiventral leaves with adaxial mesophyll and a single layer of palisade was the pattern for C. pareira (Table 2, Fig. 3A). However, bisseriate palisade was observed in five samples, being two from Brazil (CP6 and CPb), and three from Africa (CP7, CP8, CP9) (Table 2, Fig. 5C) According to Esau (1972)Esau, K., 1972. Anatomia Vegetal. Omega, Barcelona., Levitt (1980)Levitt, J., 1980. Responses of Plants to Environmental Stresses, vol. 2. New York, Academic Press. and Rozema et al. (1997)Rozema, J., Chardonnens, A., Tosserams, M., Hafkenscheid, R., Bruijnzeel, S., 1997. Leaf thickness and UV-B absorbing pigments of plants in relation to an elevational gradient along the Blue Mountains, Jamaica. Plant Ecol. 128, 150-159., palisade and spongy parenchyma are tissues known to reveal responses related to light and soil water variations. All samples showed 3–5-layered spongy parenchyma with large intercellular spaces (Fig. 3A), and several collateral vascular bundles distributed throughout the mesophyll.

The leaf margin was slightly curved toward the abaxial surface with a many-layered collenchyma and a single vascular bundle. The dorsiventral organization of the leaf mesophyll is characteristic to Cissampelos, according to Metcalfe and Chalk (1979)Metcalfe, C.R., Chalk, L., 1979. Anatomy of the Dicotyledons: Systematic Anatomy of Leaf and Stem, with a Brief History of the Subject, vol. 1., 2nd ed. Claredon Press, Oxford/United Kingdom, pp. 267. and De Wet et al. (2002)De Wet, H., Tilney, P.M., Van Wyk, B.E., 2002. Vegetative morphology and anatomy of Cissampelos in South Africa. S. Afr. J. Bot. 68, 181-190., and does not constitute an exclusive character of C. pareira.

The midrib was biconvex, in transverse section, more prominent and rounded to the abaxial surface with a sub-epidermal 3–5-layered lacunar collenchyma, followed by the fundamental parenchyma. The vascular system has a single vascular bundle with a sclerenchymatous ring at the middle portion, and 1–2 smaller vascular bundles at the base and apex (Table 2, Fig. 3B). Five samples, three from Africa (CP7, CP8 and CP9) and two from Brazil (CP6 and CPb) showed midrib with plane-convex shape, differing from the more common biconvex pattern characteristic of C. pareira (Table 2, Figs. 4D and 5D). In our studies, the morphology of the midrib in Cissampelos and other genera of Menispermaceae (in prep.), constitutes an important feature to separate taxa at specific level, which is also corroborated by studies of De Wet et al. (2002)De Wet, H., Tilney, P.M., Van Wyk, B.E., 2002. Vegetative morphology and anatomy of Cissampelos in South Africa. S. Afr. J. Bot. 68, 181-190..

In transverse section, the petiole of C. pareira showed circular contour with uniseriate epidermis and simple long trichomes; the cortex under the epidermis has continuous collenchyma similar to the midrib; the vascular system is formed by 6–8 collateral vascular bundles (Fig. 3CE), which are surrounded by a sclerenchymatic ring at the middle portion (Table 2, Fig. 3D). Six samples (CP3, CP5, CP6, CPa, CPb and CPc) showed 9–12 collateral vascular bundles (Table 2, Fig. 4E), thus differing from the pattern observed for C. pareira and therefore suggesting these samples belong to different taxa.

According to Sinnot (1914)Sinnot, E.W., 1914. The anatomy of the node as an aid in the classification of angiosperms. Am. J. Bot. 7, 303-322., Metcalfe and Chalk (1950)Metcalfe, C.R., Chalk, L., 1950. Anatomy of Dicotyledons: Leaves, Stem, and Woods in Relation to Taxonomy with Notes on Economic Uses, vol. 1. Clarendon Press, Oxford, United Kingdom, pp. 52–62. and Howard (1979)Howard, R., 1979. The Petiole. In: Metcalfe, C.R., Chalk, L. (Eds.), Anatomy of the Dicotyledons, 1. Clarendon Press, Oxford, pp. 88–96., the characteristics of the angiosperms have great taxonomic significance because the environment does not influence it. Moreover, the petiole vascularization is diagnostic to distinguish some genera and species and also has taxonomic importance (Wilkinson, 1978Wilkinson, P., 1978. Leaf anatomy of the Tribe Coscinieae Hook. f. & Thoms. (Menispermaceae). Kew Bull. 32, 347-360., 1986Wilkinson, H.P., 1986. Leaf anatomy of Tinomiscium and Fibraurea (Menispermaceae tribe Fibraureeae) with special reference to laticifers and astrosclereids. Kew Bull. 41, 153-169., 1989Wilkinson, H.P., 1989. Leaf anatomy of the Menispermaceae tribe Tiliacoreae Miers. Bot. J. Linn. Soc. 99, 125-174.). On the other hand, the sclerenchymatic sheath was also referred to C. sympodialis by Porto et al. (2008)Porto, N.M., Basílio, I.J.L.D., Agra, M.F., 2008. Pharmacobotanical study of the leaves of Cissampelos sympodialis Eichl., (Menispermaceae). Braz. J. Pharmacogn. 18, 102-107., and to other South African Cissampelos species by De Wet et al. (2002)De Wet, H., Tilney, P.M., Van Wyk, B.E., 2002. Vegetative morphology and anatomy of Cissampelos in South Africa. S. Afr. J. Bot. 68, 181-190.. It is common in vegetative organs of Menispermaceae, especially in lianas (Carlquist, 1996Carlquist, S., 1996. Wood and stem anatomy of Menispermaceae. Aliso 14, 155-170.).

Idioblasts were observed in the vascular tissue of the cortical parenchyma of the midrib and petiole and showed a positive reaction to alkaloids in all samples of C. pareira (Table 2, Fig. 3F). Differently, the samples CP3, CP5, CP6, CP8, CP9, CPa, CPb and CP5 showed no positive reaction to alkaloids (Fig. 5G and H). These results corroborate with existing chemical studies of Cissampelos (Semwal et al., 2014Semwal, D.K., Semwal, R.B., Vermaak, I., Viljoen, A., 2014. From arrow poison to herbal medicine: the ethnobotanical, phytochemical and pharmacological significance of Cissampelos (Menispermaceae). J. Ethnopharmacol. 155, 1011-1028.), as well as confirm the localization of the production and/or accumulation of these compounds in the leaf (Chowdhury, 1972Chowdhury, A.R., 1972. Chemical investigations on Cissampelos pareira. Sci. Cult. 38, 358.; Cavalcanti et al., 2014Cavalcanti, A.C., Gomes, A.N.P., Porto, N.M., Agra, M.F., Moura, T.F.A.L., Oliveira, E.J., 2014. Phamacognostic evaluation of Cissampelos sympodialis Eichl leaves. S. Afr. J. Bot. 93, 70-78.). According to Menachery (1996)Menachery, M.D. 1996. The alkaloids of South American Menispermaceae. In S.W. Pelletier (org), Alkaloids: Chemical and Biological Perspectives. New York: Ed. Pergamon, p 269–302. and Barbosa-Filho et al. (1997)Barbosa-Filho, J.M., Agra, M.F., Thomas, G., 1997. Botanical, chemical and pharmacological investigation on Cissampelos species from Paraíba (Brazil). Sci. Cult. 49, 386-394., the occurrence of alkaloids can be a chemotaxonomic character of Menispermaceae.

Secretory cavities were observed at the leaf apex of two samples from Africa (CP8 and CP9), and also in the midrib and petiole of eight samples (Table 2, Figs. 4H and 5E, F), three from Africa (CP7, CP8 and CP9), and six from Brazil (CP3, CP5, CP6, CPa, CPb and CPc). Although this character has been reported for some species of Cissampelos and other genera of Menispermaceae (Metcalfe and Chalk, 1950Metcalfe, C.R., Chalk, L., 1950. Anatomy of Dicotyledons: Leaves, Stem, and Woods in Relation to Taxonomy with Notes on Economic Uses, vol. 1. Clarendon Press, Oxford, United Kingdom, pp. 52–62., 1979Metcalfe, C.R., Chalk, L., 1979. Anatomy of the Dicotyledons: Systematic Anatomy of Leaf and Stem, with a Brief History of the Subject, vol. 1., 2nd ed. Claredon Press, Oxford/United Kingdom, pp. 267.; Wilkinson, 1989Wilkinson, H.P., 1989. Leaf anatomy of the Menispermaceae tribe Tiliacoreae Miers. Bot. J. Linn. Soc. 99, 125-174.; De Wet et al., 2002De Wet, H., Tilney, P.M., Van Wyk, B.E., 2002. Vegetative morphology and anatomy of Cissampelos in South Africa. S. Afr. J. Bot. 68, 181-190.), it was never mentioned before to C. pareira, and provides further evidence that these samples with secretory cavities belong to different taxa. According to Fahn (1988)Fahn, A., 1988. Secretory tissues in vascular plants. New Phytol. 108, 229-257., secretory cavities are distinctive for angiosperms and have important taxonomic value.

The results of the leaf anatomy of thirteen samples analyzed identified as C. pareira, revealed that only five of these samples (CP1, CP2, CP4, CPd and CPe), all from Brazil, showed a set of characters that match the pattern described for C. pareira by Porto et al. (2011)Porto, N.M., Figueiredo, R.C.B.Q., Oliveira, A.F.M., Agra, M.F., 2011. Leaf epidermal characteristics of Cissampelos L. (Menispermaceae) species from Northeastern Brazil. Microsc. Res. Tech. 74, 370-376. and Sudhakaran (2012)Sudhakaran, M.V., 2012. Histo-morphological, fluorescent and powder microscopic characterization of Cissampelos pareira Linn. Phcog. J. 4, 57-68., which are summarized here: hypostomatic leaf blades; anomocytic and anisocytic stomata with predominance of the first type at the epidermis level; epidermal anticlinal walls curves on the adaxial surface, and wavy on the abaxial surface; epidermis with thin cuticle and simple uniseriate trichomes; dorsiventral mesophyll with uniseriate palisade parenchyma, with few and rare styloid cristals (Table 2, Fig. 4H); biconvex midrib; collateral vascular system; petiole with 6–7 vascular bundles; idioblasts positive for alkaloids; and no secretory cavities.

Nine samples (CP3, CP5, CP6, CP7, CP8, CP9, CPa, CPb and CPc) were mistakenly identified as C. pareira, as they have a set of characters different of the pattern of C. pareira. These set of different characters displayed by these samples allow to separate them into three groups, which could belong to distinct and unidentified taxa (Table 2): Cissampelos sp1, CP3, CP5, CPa and CPc (Fig. 4A and B); Cissampelos sp2 (Fig. 4CH), CP6 and CPb; and Cissampelos sp3, CP7, CP8 and CP9 (Fig. 5AH).

Cissampelos sp1 and Cissampelos sp3 have secretory cavities, biconvex midrib, and epidermal papillae on the abaxial surface. However, they present distinctive characters: Cissampelos sp1 has uniseriate palisade parenchyma, and the petiole with 9–12 vascular bundles (Table 2); while in Cissampelos sp3 the palisade parenchyma is biseriate and the petiole has 6–8 vascular bundles.

On the other hand, samples of Cissampelos sp2 differs from Cissampelos sp1 and Cissampelos sp3 mainly by the anticlinal walls of epidermal cells that are straight and smooth with thin cuticle (Fig. 4C and D), without papillae. However, it shows a biseriate mesophyll and plane-convex midrib, similar to Cissampelos sp3, but different from Cissampelos sp1 that has an uniseriate palisade, and biconvex midrib. The number of petiole vascular bundles of Cissampelos sp2 is similar to that showed by Cissampelos sp1 (9–12), but different from Cissampelos sp3, with 6–8 vascular bundles.

The evidences found in this work suggest that nine samples were mistakenly identified as C. pareira, and probably belong to three different taxa, as they have a distinctive set of characters from those observed C. pareira. With regard to identification of these indeterminate taxa, further investigations are required with a higher sampling allowing for the identification of the taxa involved.

Spectroscopy analysis and principal component analysis (PCA)

The spectral profile showed saturated absorbance in the characteristic region of conjugated enones (240–190 nm) while the region above 550 nm showed no relevant absorbance, only bands of possible interferents, related to chlorophyll and/or other substances (Shipman et al., 1976Shipman, L.L., Cotton, T.M., Norris, J.R., Katz, J.J., 1976. An analysis of the visible absorption spectrum of chlorophyll a monomer, dimer, and oligomers in solution. J. Am. Chem. Soc. 98, 8222-8230.). These results led us to select the spectral range from 550 to 250 nm (600 variables) for chemometric studies (Fig. 6).

Fig. 6
Cissampelos species: UV–vis spectra of in the spectral region selected for chemometric analysis (550–250 nm). For the numerical code of the species see Table 1.

The characterization of spectra was carried out by the analysis of methanol extracts of specimens. Four distinct absorption bands were observed (Table 3): Band I (λmax 500–504.5 nm); Band II (λmax 391.5–468 nm); Band III (λmax 312.5–368.5 nm); Band IV (λmax 223–291 nm).

Table 3
Maximum absorption values (λmax) for Cissampelos species and out-group.

In the interspecific analysis all analyzed species showed similar absorption for the bands I and III, which may be related to the overlap of absorption in this region. However, in the region of band II (λmax at 403.5–410.0 nm), Cissampelos tropaeolifolia showed significantly different absorption values at λmax 466.5 nm, when compared with other species.

The intraspecific analysis of samples of C. pareira revealed shifts in the wavelength of maximum absorption in the band IV between Brazilian (λmax at 261.5–272.0 nm) and African (λmax at 282.0–284.0 nm) specimens, except for the sample CP9 that showed no band. The spectral analysis of C. pareira samples from Brazil revealed absorption maxima at 261.5 and 272 nm, which are characteristic of the C=N chromophore (Nagarajan et al., 2011Nagarajan, K., Chauhan, N., Mittal, A., Singh, V., Bodla, R.B., Tiwari, R.K., 2011. Phytochemical extraction, optimization and physico-chemical characterization of two bioactive isolates from the leaves and stem of Cissampelos pareira. Der Pharma Chem. 3, 327-337.). Based on the data available, such absorbance can be related to the presence of tropoisoquinoline alkaloids, which were already isolated from C. pareira (Morita et al., 1993Morita, H., Matsumoto, K., Takeya, K., Itokawa, H., Iitaka, Y., 1993. A novel antileukemic tropoloisoquinoline alkaloid, pareirubrine, from Cissampelos pareira. Chem. Lett., 339-342.), and is also common in other species of Cissampelos (Menachery, 1996Menachery, M.D. 1996. The alkaloids of South American Menispermaceae. In S.W. Pelletier (org), Alkaloids: Chemical and Biological Perspectives. New York: Ed. Pergamon, p 269–302.).

The spectral data of the outgroup samples, A. chloranthum, A. steyermarkii, H. domingensis, O. hirsuta, O. schomburgkii, S. brachypoda were different, as expected, from C. pareira and other species of the genus, especially C. andromorpha, C. sympodialis and C. tropaeolifolia in the PC1 factor (Fig. 7A).

Fig. 7
Cissampelos species (A) PC1 × PC2; (B) PC1 × PC3: PCA spectral profile (770–250 nm).

The principal component analysis of spectral data allowed the differentiation of samples of C. pareira of African and Brazilian origin mainly based on their score values at PC3. The spectra of Brazilian samples CP4 and CP6 showed proximity with the samples of the African group, CP7, CP8 and CP9 (Fig. 8). The samples of C. pareira specimens were close to C. tropaeolifolia and C. sympodialis with positive score values in the PC3 (Fig. 7B). It is worth noting a group of African (CP7, CP8 and CP9) and Brazilian samples (CP4 and CP6) that, although similar, has spread throughout the dispersion diagram (Fig. 8). Based on the results, in general, the PCA analysis was able to delimit the species of Cissampelos analyzed.

Fig. 8
Cissampelos pareira and relatives from Brazil and Africa (PC1 × PC2): PCA spectral profile (770–250 nm).

The results showed that application of UV/Vis spectrophotometry is a valid technique for identifying Cissampelos samples, confirming the observations already made for other angiosperms groups such as Asteraceae by Lonni et al. (2005)Lonni, A.A.S.G., Scarminio, I.S., Silva, L.M.C., Ferreira, D.T., 2005. Numerical taxonomic characterization of Baccharis genus species by ultravioleta-visible spectrophotometry. Anal. Sci. 21, 235-239. and Solanum by Basílio et al. (2012)Basílio, I.J.L.D., Bhattacharyya, J., Moura, R.K.P., Agra, M.F., 2012. Application of UV/VIS spectrophotometry and multivariate analysis to characterization of the species of Solanum sect. Erythrotrichum CHILD. Chem. Biodivers. 9, 1114-1124..

The chemical data corroborates with the anatomical features of C. pareira samples, which differentiates them from samples of other species with the following characters: biseriate parenchyma, stomata above the level of the epidermis, presence of epidermal papillae as well as secretory cavities in the mesophyll.

The principal components analysis (PCA) provided important spectral information, which allowed the separation of African-tropical and Neotropical samples. These differences may be related to geographical distribution, considered by Griffin and Lin (2000)Griffin, W.J., Lin, G.D., 2000. Chemotaxonomy and geographical distribution of tropane alkaloids. Phytochemistry 53, 623-637. as an aspect that influences the chemical composition of plants. However, when analyzing chemical data together with anatomical characters, usually neglected in taxonomic studies, these differences suggest that the studied samples belong to different taxa, probably in speciation, after being subjected to different selection processes, with the development of adaptive characters.

Conclusions

Characters of epidermal cells, mesophyll (palisade parenchyma layers), midrib shape, number of vascular bundles in petiole, secretory cavities, and the chemical results proved to be useful and distinctive to separate C. pareira from related species, providing an additional tool for its taxonomy, and quality control of their drugs composed by leaves. Further chemical and taxonomic studies are needed to enable the identification of all taxa, as well as to explain some variations in spectral and anatomical data to C. pareira and its related species.

Acknowledgments

The authors are grateful to CAPES for financial support, and CNPq for the scholarships to NM Porto and MF Agra. The authors thank to Dr. Eduardo de Jesus Oliveira for the English revision and suggestions, and Dr. Rosa Ortiz for her valuable comments and help with the bibliography; to Missouri Botanical Garden for the institutional support; the curators of herbaria CEPEC, IAN, JPB, MG, RB, UFP and MO for their pleasant cooperation during our visits and loan of plant material; and Dulce Gonçalves for her technical support.

References

  • Abbasi, A.M., Khan, M.A., Ahmad, M., Zafar, M., Jahan, S., Sultana, S., 2010. Ethnopharmacological application of medicinal plants to cure skin diseases and in folk cosmetics among the tribal communities of North-West Frontier Province, Pakistan. J. Ethnopharmacol. 128, 322-335.
  • Ahmad, R., Malik, M.A., Zia-ul-Haq, M., 1992. Alkaloids of Cissampelos pareira Fitoterapia 63, 282-285.
  • Amresh, G., Zeashan, H., Rao, C.V., Singh, P.N., 2007a. Prostaglandin mediated anti-inflammatory and analgesic activity of Cissampelos pareira Acta Pharm. Sci. 49, 153-160.
  • Amresh, G., Zeashan, H., Gupta, R.J., Kant, R., Rao, C.V., Singh, P.N., 2007b. Gastroprotective effects of ethanolic extract from Cissampelos pareira in experimental animals. J. Nat. Med. 61, 323-328.
  • Arkarapanthu, A., Chavasit, V., Sungpuag, P., Phuphathanaphong, L., 2005. Gel extracted from Khruea-ma-noi (Cyclea barbata Miers) leaves: chemical composition and gelation properties. J. Sci. Food Agric. 85, 1741-1749.
  • Barbosa-Filho, J.M., Agra, M.F., Thomas, G., 1997. Botanical, chemical and pharmacological investigation on Cissampelos species from Paraíba (Brazil). Sci. Cult. 49, 386-394.
  • Barthlott, W., Neinhuis, C., Cutler, D., Ditsch, F., Meusel, I., Theisen, I., Wilhelmi, H., 1998. Classification and terminology of plant epicuticular waxes. J. Linn. Soc. 126, 237-260.
  • Basílio, I.J.L.D., Bhattacharyya, J., Moura, R.K.P., Agra, M.F., 2012. Application of UV/VIS spectrophotometry and multivariate analysis to characterization of the species of Solanum sect. Erythrotrichum CHILD. Chem. Biodivers. 9, 1114-1124.
  • Bone, R.E., Lee, D.W., Norman, J.M., 1985. Epidermal cells functioning as lenses in leaves of tropical rainforest shade plants. Appl. Opt. 24, 1408-1412.
  • Braga, J.M.A. Menispermaceae in Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro. http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB10031 (accessed July 2015).
    » http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB10031
  • Bridson, D., Forman, L., 1999. The Herbarium Handbook, 3rd ed. Royal Botanic Gardens, Kew.
  • Bukatsch, F., 1972. Azul de Astra e Safranina. In: Kraus, J., Arduin, M. (Eds.), Manual básico de métodos em morfologia vegetal. Edur, Seropédia, Rio de Janeiro, p. 26.
  • Carlquist, S., 1996. Wood and stem anatomy of Menispermaceae. Aliso 14, 155-170.
  • Cavalcanti, A.C., Gomes, A.N.P., Porto, N.M., Agra, M.F., Moura, T.F.A.L., Oliveira, E.J., 2014. Phamacognostic evaluation of Cissampelos sympodialis Eichl leaves. S. Afr. J. Bot. 93, 70-78.
  • Chowdhury, A.R., 1972. Chemical investigations on Cissampelos pareira Sci. Cult. 38, 358.
  • De Wet, H., Tilney, P.M., Van Wyk, B.E., 2002. Vegetative morphology and anatomy of Cissampelos in South Africa. S. Afr. J. Bot. 68, 181-190.
  • Ensikat, H.J., Ditsche-Kuru, P., Neinhuis, C., Barthlott, W., 2011. Superhydrophobicity in perfection: the outstanding properties of the lotus leaf. Beilstein J. Nanotechnol. 2, 152-161.
  • Esau, K., 1972. Anatomia Vegetal. Omega, Barcelona.
  • Franklin, G.L., 1945. Preparation of thin sections of synthetic resins and wood-resin composites, and a new macerating method for wood. Nature 155, 51.
  • Fahn, A., 1988. Secretory tissues in vascular plants. New Phytol. 108, 229-257.
  • Furr, M., Mahlberg, P.G., 1981. Histochemical analyses of laticifers and glandular trichomes in Cannabis sativa J. Nat. Prod. 44, 153-159.
  • Ganguly, M., KrBorthakur, M., Devi, N., Mahanta, R., 2007. Antifertility activity of the methanolic leaf extract of Cissampelos pareira in female albino mice. J. Ethnopharmacol. 111, 688-691.
  • Griffin, W.J., Lin, G.D., 2000. Chemotaxonomy and geographical distribution of tropane alkaloids. Phytochemistry 53, 623-637.
  • Hammer, Ø., Harper, D.A.T., Ryan, P.D., 2001. PAST: Paleontological statistics software package for education and data analysis. Palaeontol. Electron. 4, 1-9.
  • Haque, M.A., Shaha, M.K., Ahmed, S.U., Akter, R., Rahman, H., Chakravotry, S., Imran, A.H.M.N., Islam, M.T., Das, R.C., Rahmatullah, M., 2011. Use of inorganic substances in folk medicinal formulations: a case study of a folk medicinal practitioner in Tangail district, Bangladesh. Am. Eurasian J. Sustain. Agric. 5, 415-423.
  • Hedberg, I., Hedbrerg, O., Madati, P.J., Mshigeni, K.E., Mshiu, E.N., Samuelsson, G., 1983. Inventory of plants used in traditional medicine in Tanzania. II. Plants of the families Dilleniaceae-Opiliaceae. J. Ethnopharmacol. 9, 105-127.
  • Hong, Y.P., Pan, K.Y., Chen, Z.D., Lu, A.M., 2001. Characters of leaf epidermis and their systematic significance in Menispermaceae. Acta Bot. Sin. 43, 615-623.
  • Hoot, S.B., Zautke, H., Harris, D.J., Crane, P.R., Neves, S.S., 2009. Phylogenetic patterns in Menispermaceae based on multiple chloroplast sequence data. Syst. Bot. 34, 44-56.
  • Howard, R., 1979. The Petiole. In: Metcalfe, C.R., Chalk, L. (Eds.), Anatomy of the Dicotyledons, 1. Clarendon Press, Oxford, pp. 88–96.
  • Jannu, V., Sai Vishal, D., Ranjith Babu, V., Harisha, B., Ravi Chandra Sekhara Reddy, D., 2011. Antidiabetic activity of hydro-alcoholic extract of Cissampelos pareira Linn. Leaves in streptozotocin induced diabetic rats. Int. J. Pharm. Technol. 3, 3601-3611.
  • Johansen, D.A., 1940. Plant Microtechnique. McGraw Hill, New York.
  • Kamble, S.Y., More, T.N., Patil, S.R., Pawar, S.G., Ram Bindurani Bodhankar, S.L., 2008. Plants used by tribes of Northwest Maharashtra for the treatment of gastrointestinal disorders. Indian J. Tradit. Knowl. 7, 321-325.
  • Kumar, V.P., Chauhan, N.S., Padh, H., Rajani, M., 2006. Search for antibacterial and antifungal agents from selected Indian medicinal plants. J. Ethnopharmacol. 107, 182-188.
  • Kupchan, S.M., Yokoyama, N., Beal, J.L., 1960a. Menispermaceae alkaloids. I. The alkaloids of Cissampelos pareira Linn. and the origin of radix pareira brave. J. Am. Pharm. Assoc. 49, 727.
  • Kupchan, S.M., Slade, P., Young, R.J., 1960b. Intramolecular catalysis. Facilitation of alkaline hydrolysis of alicyclic 1,2-diol monoesters. Tetrahedron Lett. 1, 22-25.
  • Levitt, J., 1980. Responses of Plants to Environmental Stresses, vol. 2. New York, Academic Press.
  • Lewis, W.H., Elvin-Lewis, M.P.F., 1977. Medical Botany. Wiley-Interscience, New York.
  • Lonni, A.A.S.G., Scarminio, I.S., Silva, L.M.C., Ferreira, D.T., 2005. Numerical taxonomic characterization of Baccharis genus species by ultravioleta-visible spectrophotometry. Anal. Sci. 21, 235-239.
  • Mantuano, D.G., Barros, C.F., Scarano, F.R., 2006. Leaf anatomy variation within and between three restinga populations of Erythroxylum ovalifolium Peyr. (Erythroxylaceae) in Southeast Brazil. Rev. Bras. Bot. 29, 209-215.
  • Menachery, M.D. 1996. The alkaloids of South American Menispermaceae. In S.W. Pelletier (org), Alkaloids: Chemical and Biological Perspectives. New York: Ed. Pergamon, p 269–302.
  • Metcalfe, C.R., Chalk, L., 1950. Anatomy of Dicotyledons: Leaves, Stem, and Woods in Relation to Taxonomy with Notes on Economic Uses, vol. 1. Clarendon Press, Oxford, United Kingdom, pp. 52–62.
  • Metcalfe, C.R., Chalk, L., 1979. Anatomy of the Dicotyledons: Systematic Anatomy of Leaf and Stem, with a Brief History of the Subject, vol. 1., 2nd ed. Claredon Press, Oxford/United Kingdom, pp. 267.
  • Morita, H., Matsumoto, K., Takeya, K., Itokawa, H., Iitaka, Y., 1993. A novel antileukemic tropoloisoquinoline alkaloid, pareirubrine, from Cissampelos pareira Chem. Lett., 339-342.
  • Nagarajan, K., Chauhan, N., Mittal, A., Singh, V., Bodla, R.B., Tiwari, R.K., 2011. Phytochemical extraction, optimization and physico-chemical characterization of two bioactive isolates from the leaves and stem of Cissampelos pareira Der Pharma Chem. 3, 327-337.
  • NAPRALERT, Natural Products Alert. http://www.napralert.org/ (accessed March 2013).
    » http://www.napralert.org/
  • Nurit-Silva, K., Basílio, I.J.L.D., Agra, M.F., 2007. Estudo farmacobotânico comparativo entre Solanum paniculatum L. e Solanum rhytidoandrum Sendtn. Rev. Bras. Biol. 5, 243-245.
  • Nurit-Silva, K., Agra, M.F., 2011. Leaf epidermal characters of Solanum sect. polytrichum (Solanaceae) as taxonomic evidence. Microsc. Res. Tech. 74, 1186-1191.
  • Ortiz, R., 2001. Menispermaceae. In Stevens, W.D., Ulloa Ulloa, C., Pool, A., Montiel, O. M. (orgs.), Flora de Nicaragua. Missouri: Monographs in Systematic Botany from the Missouri Botanical Garden, p. 1432–1442.
  • Porto, N.M., Basílio, I.J.L.D., Agra, M.F., 2008. Pharmacobotanical study of the leaves of Cissampelos sympodialis Eichl., (Menispermaceae). Braz. J. Pharmacogn. 18, 102-107.
  • Porto, N.M., Figueiredo, R.C.B.Q., Oliveira, A.F.M., Agra, M.F., 2011. Leaf epidermal characteristics of Cissampelos L. (Menispermaceae) species from Northeastern Brazil. Microsc. Res. Tech. 74, 370-376.
  • Porto, N.M., Araújo, N.D., Basílio, I.J.L.D., Agra, M.F., 2012. Analysis of leaf epidermal characters of medicinal and poisonous Brazilian Menispermaceae. Planta Med. 78, 1111.
  • Priya, G., Saravanan, K., Renuka, C., 2012. Medicinal plants with potential antifertility activity – a review of sixteen years of herbal medicine research (1994–2010). Int. J. Pharm. Tech. Res. 4, 481-494.
  • Ramasubramaniaraja, R., Babu, N.M., 2010. Antihelminthic studies and medicinal herbs – an overview. Int. J. Pharm. Sci. Rev. Res. 5, 39-47.
  • Ramirez, I., Carabot, A., Melendez, P., Carmona, J., Jimenez, M., Patel, A.V., Crab, T.A., Blunden, G., Cary, P.D., Croft, S.L., Costa, M., 2003. Cissampeloflavone, a chalcone–flavone dimer from Cissampelos pareira . Phytochemistry 64, 645-647.
  • Rhodes, D.G., 1975. A revision of the genus Cissampelos Phytologia 30, 415-485.
  • Rôças, G., Scarano, F.R., Barros, C.F., 2001. Leaf anatomical variation in Alchornea triplinervia (Spreng.) Müll. Arg. (Euphorbiaceae) under distinct light and soil water regimes. Bot. J. Linn. Soc. 136, 1-8.
  • Rozema, J., Chardonnens, A., Tosserams, M., Hafkenscheid, R., Bruijnzeel, S., 1997. Leaf thickness and UV-B absorbing pigments of plants in relation to an elevational gradient along the Blue Mountains, Jamaica. Plant Ecol. 128, 150-159.
  • Rukunga, G.M., Gathirwa, J.W., Omar, S.A., Muregi, F.W., Muthaura, C.N., Kirira, P.G., Mungai, G.M., Kofi-Tsekpo, W.M., 2009. Anti-plasmodial activity of the extracts of some Kenyan medicinal plants. J. Ethnopharmacol. 121, 282-285.
  • Rury, P.M., 1983. Pareira Brava: 19th Century notes and commercial samples from E. R. Squibb M. D.. Bot. Mus. Leaf. 29, 27-48.
  • Sampaio, V.S., Araújo, N.D., Agra, M.F., 2014. Characters of leaf epidermis of Solanum of the Brevantherum Clade from Atlantic Forest of Northeastern Brazil. S. Afr. J. Bot. 74, 108-113.
  • Sanchez Medina, A., Garcia Sosa, K., May Pat, F., Pena Rodriguez, L.M., 2001. Antioxidant, antimicrobial and beta-glucosidase inhibition activities. Phytomedicine 8, 144-151.
  • Schmelzer, G.H., Gurib-Fakim, A. (Eds.), 2008. Plant Resources of Tropical Africa 11(1). Medicinal Plants 1. PROTA Foundation/Backhuys Publishers/CTA, Wageningen, Netherlands/Leiden, Netherlands/Wageningen, Netherlands, 791 pp.
  • Semwal, D.K., Semwal, R.B., Vermaak, I., Viljoen, A., 2014. From arrow poison to herbal medicine: the ethnobotanical, phytochemical and pharmacological significance of Cissampelos (Menispermaceae). J. Ethnopharmacol. 155, 1011-1028.
  • Sinnot, E.W., 1914. The anatomy of the node as an aid in the classification of angiosperms. Am. J. Bot. 7, 303-322.
  • Sharma, V., 1987. Biosynthesis of hayatin. Indian J. Chem. 26, 589-591.
  • Shipman, L.L., Cotton, T.M., Norris, J.R., Katz, J.J., 1976. An analysis of the visible absorption spectrum of chlorophyll a monomer, dimer, and oligomers in solution. J. Am. Chem. Soc. 98, 8222-8230.
  • Singh, V.K., Ali, Z.A., 1992. A contribution to the ethnopharmacological study of the Udaipur forests of Rajasthan, India. Fitoterapia 63, 136-144.
  • Singh, K.K., Maheshwari, J.K., 1994. Traditional phytotherapy of some plants used by the tharus of the Nainital District, Uttar Pradesh, India. Int. J. Pharmacogn. 32, 51-58.
  • Stace, C.A., 1965. Cuticular studies as an aid to plant taxonomy. Bull. Br. Mus. Nat. Hist. Bot. 4, 1-78.
  • Singthong, J., Cui, S.W., Ningsanond, S., Douglas Goff, H., 2004. Structural characterization, degree of esterification and some gelling properties of Krueo Ma Noy (Cissampelos pareira) pectin. Carbohydr. Polym. 58, 391-400.
  • Sudhakaran, M.V., 2012. Histo-morphological, fluorescent and powder microscopic characterization of Cissampelos pareira Linn. Phcog. J. 4, 57-68.
  • Shukla, P., Shukla, P., Gopalakrishna, B., 2012. Investigation of in-vitro anthelmintic activity of Cissampelos pareira Linn against Pheretima posthuma Int. J. Pharm. Sci. Res. 3, 265-267.
  • Thakur, P., Rana, A.C., 2013. Effect of Cissampelos pareira leaves on anxiety-like Behavior in experimental animals. J. Tradit. Complement. Med. 3, 188-193.
  • Thiers, B. (continuously updated) Index Herbariorum: A Global Directory of Public Herbaria and Associated Staff. New York Botanical Garden's Virtual Herbarium. New York Botanical Garden. Available from: http://sweetgum.nybg.org/ih/ (accessed 29.10.15).
    » http://sweetgum.nybg.org/ih/
  • Vaidya, B.G., 1988. Nighantu Adarsh, vol. 1. Chaukhambha Bharti Academy Publications, Varanasi, India, pp. 44–45.
  • Vardhanabhuti, B., Ikeda, S., 2006. Isolation and characterization of hydrocolloids from monoi (Cissampelos pareira) leaves. Food Hydrocolloids 20, 885-891.
  • Yadav, K.S., Yadav, N.P., Shanker, K., Thomas, S.C., Srivastav, S., Srivastava, S., Kumar Rai, V., Mishra, N., Sinha, P., 2013. Assessment of antidiabetic potential of Cissampelos pareira leaf extract in streptozotocinenicotinamide induced diabetic mice. J. Pharm. Res. 6, 874-878.
  • Wilkinson, P., 1978. Leaf anatomy of the Tribe Coscinieae Hook. f. & Thoms. (Menispermaceae). Kew Bull. 32, 347-360.
  • Wilkinson, H.P., 1979. Plant surface. In: Metcalfe, C.R., Chalk, L. (Eds.), Anatomy ofthe Dicotyledon. Claredon Press, Oxford, pp. 97–162.
  • Wilkinson, H.P., 1986. Leaf anatomy of Tinomiscium and Fibraurea (Menispermaceae tribe Fibraureeae) with special reference to laticifers and astrosclereids. Kew Bull. 41, 153-169.
  • Wilkinson, H.P., 1989. Leaf anatomy of the Menispermaceae tribe Tiliacoreae Miers. Bot. J. Linn. Soc. 99, 125-174.

Publication Dates

  • Publication in this collection
    Mar-Apr 2016

History

  • Received
    18 May 2015
  • Accepted
    20 Oct 2015
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