Guettarda calyptrata
A. Rich., originally from Cuba and known as “Contraguao”, has traditionally been used to counteract quickly and effectively burns produced by
Comocladia dentate
Jacq (Guao), vegetable species that secretes a highly caustic latex for skin and mucous membranes. A burn is a traumatic injury resulting in local and systemic injury with oxidative changes; in this sense the antioxidants play an important role. In order to offer aspects related to the quality and effectiveness of the plant, its pharmacognostic, phytochemical and antioxidant activity is presented. The morphoanatomical evaluation was carried out, physical-chemical parameters were determined for the crude drug and for the aqueous extract. The chemical profile of the extract was estimated by thin layer chromatography, ultraviolet-visible and
quantification of total phenols by Folin-Ciocalteu and total flavonoids by the colorimetric method of aluminum trichloride (AlCl
3
). Finally, the antioxidant activity was tested by the FRAP and DPPH techniques. Through the pharmacognostic study, the quality specifications of the drug and the extract were established. The methods of analysis used for the chemical profile suggested the presence of flavonoids and phenols in general. The aqueous extract showed antioxidant properties by the two methods evaluated. The study of
G. calyptrata
provided pharmacognostic, phytochemical and effectiveness as an antioxidant evidence, aspects to consider in the possible use of the plant by our Natural and Traditional Medicine.
<em>Guettarda calyptrata</em> Pharmacognostic Study Phytochemical Antioxidant Activity1. Introduction
G. calyptrata, which belongs to the botanical family of Rubiacea, is characterized by the production of bioactive metabolites (iridoids, alkaloids, antraquinones, triterpenes, saponins, etc.) with great pharmacological potential. These metabolites can be used as chemotaxonomic markers even for genera and subfamilies [1] [2] . G. calyptrate is a shrub or tree endemic to Cuba, common in all the provinces, savannahs and stony, arid and cuabales lands. It reaches a size of up to 6 m in height; presents coriaceous leaves oblong to oval or round-ovate, obtuse at the apex and heart-shaped at the base. Figure 1 shows the macromorphological characteristics of the leaves. The plant is known by the common names of contraguao, guayabillo, leather of green leaves. Traditionally, it is attributed to the leaves and barks (decoction of 2 to 3 times a day, on the skin), the medicinal property of relieving or curing burns produced by Comocladia dentata Jacq., plant that is characterized by secreting highly caustic latex from the skin and mucous membranes [3] [4] .
The natural antioxidants present in plants have gained great interest in recent decades, since oxidative stress (an unbalance between oxidants and pro-oxidants) is implicated in a large number of health conditions. It has been demonstrated that burned is a traumatic wound that result in a local and systematic damage with oxidative changes. This kind of lesion increases the xanthine-oxidase enzyme and byproducts of Lipidic peroxidation. It has been demonstrated that substances with antioxidant properties are effective in the treatment of burns, among them, antioxidants from natural fonts [5] .
There is not any evidence in scientific literature of studies related with this medicinal plant, for that purpose, the aim of this research work was to evaluate the pharmacognostic, phytochemical and antioxidant activity of G. calyptratato known necessary aspects in the development of its monograph and to support its medicinal use.
2. Materials and Methods2.1. Sample Collection and Processing
The plant was collected in November 2016 in shore area of Cojímar, Habana del Este municipality, Havana, Cuba. Plants were in phonologic state and were harbored an identified at Johannes Bisse herbarium in National Botany Garden where a voucher specimen (HFC-089021) was deposited. Only leaves of the plant were used in this research.
The leaves were dried at 40˚C in an oven model AISET model YLD-6000 (China) using 100 g of each sample per replica, determining the loss of weight and the time of dried (every 12 hours) according to NRSP 309, 1992; Miranda and Cuéllar, 2000 [6] [7] .
Macromorphological characters of the 100 leaves like leaf shape, size, color, texture, margin type, apex, base and petiole size, flower color and length etc were observed. Measurements were carried out using line ruler and a Stereoscopic microscope NTB-2B with camera model HDCE-50B (China) [7] .
2.2.2. Microscopic Analysis
Microscopic analysis was carried out on the powdered sample using a light microscope NOVEL (China) with 10× microscope objective lens, and coupled to HDCE-50B digital camera (China) and Scope Image Dynamic Pro software. Ground powder was cleared for some minutes in sodium hypochlorite solution. It was washed in water and then coloured with saffranin at 1% and stained in glycerinated gelatin according to Gattuso M and Gattuso S, 1999; and Miranda and Cuéllar, 2000 [7] [8] .
2.2.3. Physicochemical Analysis
Physicochemical analyses were carried out on the powdered sample following standard methods. Moisture content, alcohol extractive values at 30%, 50% and 80%), water extractive value and total ash, water soluble ash and acid insoluble ash value were tested for using a MUFFLE FURNACE SX2-12TP (China) [6] [8] [9] [10] .
2.2.4. Extract Preparation
The extracts were prepared with the ground material (20 g × 100 mL of water) getting the physico-chemical parameters like organoleptic properties (odor and color), pH, refraction index, relative density and total solids [7] [11] .
2.2.5. Phytochemical Profile of the Extract
1) TCL
TLCP (thin-layer chromatography plate) on silica gel with fluorescent indicator 254 nm on aluminum cards (layer thickness 0.2 mm) (10 × 20 cm) using n-butanol: acetic acid: water (BAW 65:25:10) as developing agent (v/v/v), concentrated sulfuric acid plus heat, FeCl3 and AlCl3 were the chromogenic agents. The TLCP were examined under ultraviolet (254 nm and 365 nm) and ordinary light. Vanillin at 1%, Rutin (R) and quercetin (Q) (Merck) were used as standard.
2) UV spectroscopy
The extract was analyzed on a UV-Visible spectrometer Anlytikjena Specord-200 plus model (Germany). The scan range was 200 to 700 nm.
3) Total phenolic and total flavonoid content
Total phenols was calculated by the Folin-Ciocalteu method [12] , using as reference the gallic acid (Sigma-Aldrich) at concentrations of 10, 20, 30, 40 and 50 mg/mL. On the other hands, the content of total flavonoids was carried out by the colorimetric method according to Pourmorad et al., 2006 [13] , using aluminum trichloride and quercetin (Sigma-Aldrich) as reference substance at the concentrations of 10, 15, 25, 50 and 100 μg/mL. In each case, calibration lineal curve was constructed with absorbance readied in a spectrophotometer Rayleigh UV-1601 (China) at 715 nm vs. concentration of reference compound, which was then obtained respective concentration of total content of phenol or flavonoids and SD in the studied extract (mg/mL).
2.3 Antioxidant Activity of the Extracts2.3.1. Ferric Reducing Antioxidant Power [FRAP] Assay
The Ferric Reducing Antioxidant Power (FRAP) assay was measured as described previously by Benzie and Strain (1996) [14] , which determine the ability of the sample to reduce iron ferric (Fe3+) to ferrous (Fe2+). The determinations were carried out in a spectrophotometer Rayleigh UV-1601 (China) at 593 nm. The extract was tested at 20, 30 and 40 μg/mL concentrations. The results were expressed as μmol equivalent of Vitamin C (purity 99%, Sigma-Aldrich), according to the standard curve of ascorbic acid (20, 50, 100, 400 and 800 μmol/L).
2.3.2. Free Radical Scavenging Activity
The reduction of 2,2-diphenyl-1-picrylhydracil (DPPH; Sigma-Aldrich) radical in 2,2-diphenyl-1-pryryl hydrazine was used for the antioxidant action of compounds containing -OH groups that decolorize said reagent according to Brand-Williams et al., 1995 [15] . The extract was tested at 25, 37, 5 and 50 μg/mL concentrations. The absorbance was read at 517 nm in a spectrophotometer Rayleigh UV-1601 (China) and the percentage inhibition of DPPH (% DPPH) staining was calculated by the following formula: % inhibition of the DPPH = (Abs control − Abs sample/Abs control) × 100. Experiment was carried out in triplicate and results were expressed as mean and SD.
2.4. Statistical Analysis
Results are presented as mean ± SD. Statistical analyses were performed by Student’s t-test. The values of p < 0.05 were considered significant. Duncan test was used utilizing the StatgraphicsÒ Plus, version 5.0 program. The mean effective concentration (IC50) was determined with the help of the Graphprism 5.0 statistical program.
3. Results and DiscussionBotanical Characterization of G. calyptrata
1) Macromorphological evaluation of the leaves
The macromorphological ealuation represented in Figure 2, allow the observation of decussate leaves on the stem, with aovade-eliptic shape, coreaceus texture, penninervous, short petiole (0.9 ± 0.31), cordade base, obtuse-oblongate apex and ondulate margine. Trichomes on the abxial and adaxial surfaces. Leaf length 6.79 cm (SD = 1.05) and width of 4.21 cm (SD = 0.70). Macromorpholic characteristis are according to literature data for this spice as discussed by Bisse in 1988 [3] .
2) Micromorphological evaluation of the powder drug (leaves)
Helical xylematic vessel tissue according to Gattuso M and Gattuso S, 1999 [3] , with unicellular trichomes, which were identified under macroscopic analysis. Stomata and epidermal cells were observed with lightly grossed walls, and variable size and shape (Figure 3).
3) Physicochemical parameters of the leaf powder
Table 1 summarizes the results of determination of physicochemical para meters of powered drug. Moisture content is according with the range accepted for medicinal plants. Water soluble extractive is acceptable while alcohol soluble extractives are higher than the aqueous extractive indicating that chemical compounds have a half-polarity, increasing the values with the ethanol concentration. All ashes values are beneath the permitted values, demonstrating that the drug was clean and with a low content of metals [6] [7] [9] .
4) Physicochemical Parameters of the extract
Table 2 summarizes the results of physicochemical parameters. The pH of the extract was noted to be 4.01 ± 04 (lightly acid), total solids value is low but is in correspondence with the results obtained in water soluble extractive, indicating that their chemical components are less polar or middle polar. The extraction with water was done according to popular use in the Cuban traditional medicine.
Physicochemical parameters of the leaf powder
Parameters (%)
Results ± SD
Limit value for herb (Commission, 2015)
Lou-Zhicen (1980)
Moisture content
8.00 ± 0.0
10.0
8.0 - 14.0
Water soluble extractive
10.57 ± 0.14
-
-
Alcohol soluble extractive at 30%
15.47 ± 0.07
-
-
Alcohol soluble extractive at 50%
19.29 ± 0.13
-
-
Alcohol soluble extractive at 80%
20.55 ± 0.08
-
-
Total ash content
3.26 ± 0.11
15.0
5.0
Water soluble ash
1.41 ± 0.16
8.0
-
Acid insoluble ash
1.81 ± 0.10
1.5
2.0
Physicochemical parameters of aqueous extract of G. calyptrata
Parameters
Results ± SD
pH
4.01 ± 0.04
Total Solids (%)
1.61 ± 0.05
Refraction index
1.3297 ± 0.0001
Relativedensity (g/mL)
0.9811 ± 0.0006
Refraction index and Relative density are characteristic of this spice.
5) Phytochemical Profile of the extract
The chemical components were separated in some way taking into account their polarities, suggesting the presence of phenolic compounds and among them, rutin flavonoid, on behalf of the Rf and the chromogenic agents used. The results are showed in Figure 4.
6) UV-Visible spectroscopy
Figure 5 shows the UV-Visible spectroscopy analysis of the extracts showed absorption bands between 270 and 285 nm and another one between 325 and 330 nm. This behavior could be related with the presence of phenolic compounds into the extracts, especially flavonoids, which exhibit two bands in the ultraviolet-visible region: Band I at 300 - 400 nm and band II at 200 - 285 nm according to Abad-Garcia, 2009 and Martínez et al., 2012 [16] [17] .
7) Phenol and flavonoid contents
Table 3 demonstrates that both results showed low concentration, suggesting that the increments of the value are possible with the use of hydroethanolic solvent and heating the extract.
8) Antioxidant activity of the extract
a) Ferric reducing antioxidant power (FRAP) assay
FRAP assay showed in Table 4, based on the reduction of ferric tripyridyltriazine complex to its ferrous colored form according to Afsar et al., 2018 [18] . The results were expressed as µM equivalent of ascorbic acid, the standard used for the analysis. The results displayed in table show the ability of the extract to reduce Fe3+ to Fe2+. Was visualized an intense blue color complex. The best result was obtained at the concentration of 40 μg/mL.
b) Free radical scavenging activity
The effect of antioxidants on DPPH radical scavenging was thought to be due to their hydrogen donating ability. DPPH is a stable free radical and accept an electron or hydrogen radical to become a stable diamagnetic molecule. The
Phenol and flavonoid content of aqueous extracts of G. calyptrata
Results
Compounds (mg/mL)
x ¯ ± SD
Phenol contents
2.83 ± 0.04
Flavonoid contents
1.14 ± 0.005
Antioxidant activity of the extract in the FRAP assay
Concentration (μg/mL)
(μM equivalents of ascorbic acid) ± SD
20
21.24 ± 1.75a
30
44.04 ± 1.76b
40
162.76 ± 6.16c
Values are expressed as mean ± Standard deviation (n = 3); means with superscript with different letters (a - c) are significantly (p < 0.05). Data analyzed by using one way ANOVA followed by Duncan test.
reduction capability of DPPH radicals was determine by the decrease in its absorbance at 517 nm induced by antioxidants [18] [19] .
Qualitatively was observable a change in the color of the solution from purple to yellow in ll concentrations evaluated, indicating that the higher inhibition percentage of DPPH radical was at the higher concentration evaluated (50 μg/mL). Table 5 shows the results with DPPH radical scavenging activity and IC50.
4. Conclusions
From the study, important diagnostic characters that might be useful in determining authenticity and identifying adulteration of the crude drug are observed. These are found in the in the abundant long unicellular unbranched trichomes, helical xylematic vessels and epidermal cells with lightly grossed walls. The micromorphological results of powdered drug of G. calyptrata have not been reported previously, this is an important contribution to know this spice.
An aqueous extract was elaborated using decoction method taking into account the traditional use giving by Cuban population. Physicochemical parameters to establish its quality were determined.
Phytochemical study allowed detects phenolic compounds and particularly rutin according the conditions tested. The presence of phenolic compounds in this spice has a great coincidence with those results reported for Guettarda gender, where were found flavonoid glycosides like quercetin-3-O-B-D-galactopiranoside y quercetin-3-O-B-D-glucopiranoside [20] . The results exhibit the first evidences of the preliminar chemical composition in G. calyptrata.
Natural antioxidants play an important role in front of oxidative stress, possessing antimutagenic, anticarcinogenic, and antiinflammatory and neuroprotective effects discussed by Hsu et al., 2012; Afsar et al., 2018 [18] [21] . These properties could be closely related to phenolic compounds that contain in its
Antioxidant activity of the extract in the DPPH assay
Concentration (μg/mL)
DPPH radical scavenging activity ± SD
25
40.09 ± 0.22a
37.5
42.25 ± 0.34b
50
52.92 ± 0.11c
IC50
38.41
Values are expressed as mean ± Standard deviation (n = 3); means with superscript with different letters (a - c) are significantly (p < 0.05). Data analyzed by using one way ANOVA followed by Duncan test.
chemical structures, a variable number of hydroxy groups that react with free radicals according to Csepregi et al., 2016; Sepahpour et al., 2018 [22] [23] .
According with those results, the presence of phenolic compounds and particularly rutin was detected and recognized by its antioxidant activity. Last evidence suggests that at least part of the antioxidant effect founded for the aqueous extract of G. calyptrate could be associated with this component.
Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper.
Cite this paper
Gaitén, Y.I.G., Yaque, J.G., Lizama, R.S., Torres, Z.O., González, A.F., Jiménez, J.L.M. and Monan, M. (2018) Pharmacognostical, Phytochemical and Antioxidant Evaluations of Guettarda calyptrata A. Rich. Open Access Library Journal, 5: e4921. https://doi.org/10.4236/oalib.1104921
ReferencesMartins, D. and Nunez, C.V. (2015) Secondary Metabolites from Rubiaceae Species. Molecules, 20, 13422-13495. https://doi.org/10.3390/molecules200713422Revathi, D. and Rajeswari, M. (2015) Chemical Profiling of Guettarda speciosa Linn. by GC-MS. International Journal of Emerging Technology and Advanced Engineering, 5, 114-118.árboles de Cuba, B.J. (1988) Editorial Científico-Técnica. Ciudad de La Habana, 265-275.Roig, J.T. (2014) Diccionario botánico de nombres vulgares cubanos. Tomo I. Cuarta edición. Editorial Científico-Técnica. La Habana, p. 295.Schencke, C., Salvo, J., Vasconcellos, A. and del Sol, M. (2013) Estudio Comparativo de la Cicatrización en Quemaduras con Tratamiento en Base a Miel de Ulmo (Eucryphia cordifolia) y Vitamina C oral versus Hidrogel en Cobayos (Cavia porcellus). International Journal of Morphology, 31, 839-844. https://doi.org/10.4067/S0717-95022013000300010NRSP 309. (1992) Norma Ramal. Medicamentos de origen vegetal. Droga cruda. Métodos de ensayo, 16-29.Miranda, M.M. and Cuéllar, A.C. (2000) Manual de prácticas de laboratorio. Farmacognosia y productos naturales. Ciudad Habana, 25-49, 74-79.Gattuso, M.A. and Gattuso, S.J. (1999) Manual de procedimientos para el análisis de drogas en polvo. Editorial de la Universidad Nacional de Rosario Urquiza, Argentina.Lou, Z.-C. (1980) General Control Methods for Vegetable Drugs. Comparative Study of Methods Included in Thirteen Pharmacopoeias: An Proposals on Their Internacional Unification. WHO/PHARM/80.502, 8-39.Commission, C.P. (2015) Pharmacopoeia of the People’s Republic of China. Chinese Medical Science and Technology Press, Beijing, 337.NRSP 312. (1992) Norma Ramal. Medicamentos de origen vegetal. Extractos fluidos y tinturas. Métodos de ensayo, 15-19.Chlopicka, J., Pasko, P., Gorinstein, S., Jedryas, A. and Zagrodzki, P. (2012) Total Phenolic and Total Flavonoid Content, Antioxidant Activity and Sensory Evaluation of Pseudocereal Breads. LWT-Food Science and Technology, 46, 548-555. https://doi.org/10.1016/j.lwt.2011.11.009Pourmorad, F., Hosseinimerhr, S.J. and Shahabimajd, N. (2006) Antioxidant Activity, Phenol and Flavonoid Contents of Some Selected Iranian Medicinal Plants. African Journal of Biotechnology, 5, 1142-1145.Benzie, I.F. and Strain, J.J. (1996) The Ferric Reducing Ability of Plasma (FRAP) as a Measure of “Antioxidant Power”. The FRAP Assay. Analytical Bio-chemistry, 239, 70-76. https://doi.org/10.1006/abio.1996.0292Brand-Williams, W., Cuvelier, M.E. and Berset, C. (1995) Use of Free Radical Method to Evaluate Antioxidant Activity. LWT-Food Science and Technology, 28, 25-30. https://doi.org/10.1016/S0023-6438(95)80008-5Abad-Garcia, B., Berrueta, L.A., Garmon-Lobato, S., Gallo, B. and Vicente, F. (2009) A General Analytical Strategy for the Characterization of Phenolic Compounds in Fruit Juices by High-Performance Liquid Chromatography with Diode Array Detection Coupled to Electrospray Ionization and Triple Quadrupole Mass Spectrometry. Journal of Chro-matography A, 1216, 5398-5415. https://doi.org/10.1016/j.chroma.2009.05.039Martínez, J., García, C., Durango, D. and Gil, J. (2012) Caracterización de propóleos provenientes del municipio de Caldas obtenido por dos métodos de recolección. Revista MVZ Córdoba, 17, 2861-2869. https://doi.org/10.21897/rmvz.254Afsar, T., Razak, S., Shabbir, M. and Rashid, K.M. (2018) Antioxidant Activity of Polyphenolic Compounds Isolated from Ethyl-Acetate Fraction of Acacia hydaspicaR. Parker. Chemistry Central Journal, 12, 5. https://doi.org/10.1186/s13065-018-0373-xBokhari, J., Khan, M.R., Shabbir, M., Rashid, U., Jan, S. and Zai, J.A. (2013) Evaluation of Diverse Antioxidant Activities of Galium aparine. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 102, 24-29. https://doi.org/10.1016/j.saa.2012.09.056Naressi, M.A., Manholer, D.D., Ames, F.Q., Bersani-Amado, C.A., Formagio, A.S.N., Pereira, Z.V., Da Costa, W.F., Baldoqui, D.C. and Sarragiotto, M.H. (2015) Chemical Constituents, Anti-Inflammatory, and Free-Radical Scavenging Activities of Guettarda viburnoides CHAM. & SCHLTDL. (Rubiaceae). Química Nova, 38, 932-936.Hsu, F.-L., Huang, W.-J., Wu, T.-H., Lee, M.-H., Chen, L.-C., Lu, H.-J., Hou, W.-C. and Lin, M.-H. (2012) Evaluation of Antioxidant and Free Radical Scavenging Capacities of Polyphenolics from Pods of Caesalpinia pulcherrima. International Journal of Molecular Sciences, 13, 6073-6088. https://doi.org/10.3390/ijms13056073Csepregi, K., Neugart, S., Schreiner, M. and Hideg, E. (2016) Comparative Evaluation of Total Antioxidant Capacities of Plant Polyphenols. Molecules, 21, 208. https://doi.org/10.3390/molecules21020208Sepahpour, S., Selamat, J., Abd Manap, Y.M., Khatib A and Abdull Razis, A.F. (2018) Comparative Analysis of Chemical Composition, Antioxidant Activity and Quantitative Characterization of Some Phenolic Compounds in Selected Herbs and Spices in Different Solvent Extraction Systems. Molecules, 23, 402.