Journal of Global Biosciences Peer Reviewed, Refereed, Open-Access Journal ISSN 2320-1355 Volume 10, Number 4, 2021, pp. 8659-8668 Website: www.mutagens.co.in URL: www.mutagens.co.in/jgb/vol.10/04/100412.pdf

Research Paper MORPHO-ANTOMICAL PROPERTIES AND PHYTOCHEMICAL CONSTITUENTS OF carnea JUST. OF THE FAMILY

Wahua, C., Odogwu, B. A. and Ukomadu, J. Department of Science and Biotechnology, University of Port Harcourt, Choba, P.M.B. 5323, Nigeria.

Abstract The study examined the morpho-anatomical properties and phytochemical constituents of Justicia carnea Just. This belongs to the Order of Acanthaceae family. It is an erect evergreen shrub by habit, which grows up to 250cm in height. The leaves are dark green, up to 15 to 20cm in length and 5 to 7cm in width, it is observed having plumes of tubular flowers with reddish corolla. Epidermal studies showcased paracytic stomata and multicellular uniseriate trichomes which are present on both adaxial and abaxial surfaces. Anatomical study showed a layer of epidermal cells. The hypodermis is made of 5 to 14 rolls of collenchyma, general cortex and pith dominated by parenchyma in the same pattern of occurrence in mid-ribs, petioles, stems, nodes and roots except that the number of rolls of cells varied slightly and vasculation is open type. The qualitative phytochemical investigations conducted revealed presence of following: alkaloids, flavonoids, tannins, anthraquinones, triterpenoids, cardenolide and saponins while phlobatannins and fixed oil components were absent. The quantitative chemical estimation studied showcased 4200.1mg/kg of Sodium (Na) read on atomic absorption at 589nm wavelength, 15230.1mg/kg of Potassium (K) at 766nm wavelength and 943.5mg/kg of Iron (Fe) at 248.3 wavelengths. The investigations conducted here could further be used to delimit the species. Key words: morphology, anatomy, phytochemistry, Justicia carnea. INTRODUCTION The family Acanthaceae has more than 4,000 species in the tropical and subtropical regions (1). Justicia is the largest among the genera in Acanthaceae with approximately 600 species worldwide (2). Occurrence of cystoliths in the vegetative parts in Acanthaceae is considered a characteristic feature of the family. Also starch grains in

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Creative Commons Attribution License 4.0 International License Journal of Global Biosciences Vol. 10(4), 2021 pp. 8659-8668 ISSN 2320-1355 plant parts have been observed by (3), (4), gypsum (5). Silica (6), raphides (7), (8), (9), (10) and (11) have conveniently been employed in taxonomic researches. Crystals of calcium oxalate referred as cystoliths or lithocyata have similar significance. The anatomical study of Acanthaceae particularly species of the genus Justicia is scanty (12). Justicia carnea has simple trichomes elongated multicellular and glandular capitate trichomes. Paracytic stomata were present (13). It is used as blood tonic, the vegetative parts turns deep red after boiling and could be administered for curative remedies due to the presence of relevant phytochemical constituents. Thus, the objectives are on the morpho-anatomical properties and phytochemical constituents of Justicia carnea Just. belonging to the Order Lamiales of the family Acanthaceae. MATERALS AND METHODS Geographic Location The location of the parent plant studied was Port Harcourt, Rivers, Nigeria. Morphological Studies The meter rule was used to ascertain the plant height from the root-collar to the terminal bud while leaf length from the leaf tip to the petiole base. The leaf width is measured across the leaf lamina, from one margin to another at the widest region. Micro-morphological (Epidermal) Studies Fresh leaves and young stem collected for this study were peeled following the method of (14) and subjected to alcohol solutions in the ratio of 30%, 50%, 70%, 95% and absolute alcohol respectively. The cleared epidermal layers obtained were stained with safranin for 5 minutes washed and counter stained with Alcian blue for same time interval, washed and temporarily mounted in aqueous glycerol solution. Photomicrographs were taken from good preparations. The stomatal index [S.I.] was obtained using the formula:

S 100 S. I. = X S + E 1 Where S and E are mean numbers of stomata and epidermal cells respectively within the particular area under investigation. Likewise trichome Index (T.I) was obtained using:

T 100 T. I. = X T + E 1 Where T and E are trichomes and epidermal cells respectively within the study area.

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Anatomical Study The plant was harvested from the wild for the secondary anatomy. The harvested stems, leaves, petioles, flowers, fruits and roots were dehydrated in alcohol solutions of 50%, 75%, absolute alcohol and thereafter subjected through alcohol chloroform series in the ratio of 3:1 of alcohol chloroform series, 1:1, 1:3 and pure chloroform respectively for five minutes in each. This was there after rehydrated following same procedure to 50% alcohol before staining with safranin for 2 to 5 minutes, counter stained with Alcian blue for same time interval. Free hand section was done using a systematic arrangement of 5 razor blades as described by (15) was also adopted. Microphotographs were taken from good preparations using Sony camera of 7.2 Mega pixels having 2.411 LCD monitor and High sensitivity ISO 1250. Qualitative Phytochemical Study Leaves of Justicia carnea studied were sun dried for 72 hours (3 days) and weighed. Fifty grammes (50g) of the dried leaves were macerated in 96% ethanol using a pestle and a mortar. The extract was thereafter filtered and evaporated to dryness (constant weight) using a rotary evaporator set at 450C. Residue yields were noted and a portion was used for the phytochemical screening. Test for Saponins Frothing tests was done following the method described by (16). The ability of saponins to produce frothing in aqueous solution and to haemolyse red blood cells was used as screening test for these compounds. 0.5g of the plant extract was shaken with water in a test tube. Frothing which persisted on warming was taken as preliminary evidence for the presence of saponins. The disc was then washed in ether, dried and placed on a 7% blood nutrient agar. Complete haemolysis of red blood cells around the disc after 6 hours was taken as further evidence of presence of saponins. Test for alkaloids This was carried out using 0.5g of the plant extract which was stirred with 5ml of 1% aqueous hydrochloric acid on a steam bath; 1ml of the filtrate was treated with a few drops of Mayer’s reagent and a second 1ml portion was treated similarly with Dragendorff’s reagent. Turbidity or precipitation with either of these reagents was taken as preliminary evidence for the presence of alkaloids in the extract being evaluated (17) and (18). A modified form of the tin-layer chromatography (TLC) method as described by (19) was used. One gramme (1g) of the extract was treated with

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40% calcium hydroxide solution until the extract was distinctly alkaline to Litmus paper, and then extracted twice with 10ml portions of chloroform. The extracts were combined and concentrated to 5mls. The chloroform extract was then spotted on thin- layer plates. Four different solvent systems were used to develop each plant extract. The presence of alkaloids in the developed chromatograms was detected by spraying the chromatograms with freshly prepared Dragendorff’s spray reagent. A positive reaction on the chromatograms (indicated by an orange or darker colored spot against a pale yellow background) was confirmatory evidence that the plant extract contained alkaloid. Test for tannins Five grammes (5g) of each portion of plant extract was stirred with 10ml of distilled water, filtered, and 5% ferric chloride reagent added to the filtrate. A blue-black, green, or blue-green precipitate was taken as evidence for the presence of tannins (18). Test for anthraquinones Borntrager’s test was used. Five grammes (5g) of each plant extract was shaken with 10ml benzene, filtered and 5mls of 10% ammonia solution added to the filtrate. The mixture was shaken and the presence of a pink, red, or violet color in the ammonia (lower) phase indicated the presence of free hydroxyanthraquinones Test for phlobatannins The deposition of a red precipitate when an aqueous extract of the plant part was boiled with 1% aqueous hydrochloric acid was taken as evidence for the presence of phlobatannins (18). Test for flavonoids Shinoda reduction test: 5g of the pulverized sample was boiled in 5ml of distilled water for 5 minutes on water bath and filtered while hot. Magnesium (Mg) was added to the filtrate and few drops of conc.H2SO4 were carefully introduced into the mixture. The formation of orange, red, crimson or magenta was taken as evidence of preliminary presence of flavonoid. Lead acetate test: 5g of pulverized sample was boiled in 5ml of distilled water for 5 minutes in water bath and filtered while hot. 2ml of 10% lead acetate was added to the filtrate and observed. Yellow precipitate indicated presence of flavonoids.

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Test for cardiac glycosides Lieberman’s test was used in which 0.5g of the extract was dissolved in 2ml of acetic anhydride and cooled in ice. One milliliter (1ml) of Sulphuric acid was carefully added in drops until a color change from violet to blue to green indicated the presence of a steroidal aglycone portion of the cardiac glycoside (20). Steroids and Terpenoids Salkowski’s Test: 2g of plant sample was pulverized and macerated in 5mls of chloroform and filtered. 2mls of H2SO4 was carefully added to the filtrate and observed. A reddish brown colour at the interface indicated presence of steroidal substances. Libermann-Burchard’s Test: 2g of plant sample was pulverized and macerated in 5mls of chloroform and filtered. 1ml of acetic anhydride was added to the filtrate followed by

2mls of conc.H2SO4 to form a layer. Color change from violet to blue to green at interface showed the presence of terpenoids. Fixed Oil About 5 grammes of plant sample was vigorously but carefully rubbed with filter paper. The paper was allowed to air dry for about 5 to 10 minutes and observed. Observation of translucent coloration revealed the presence of fixed oil. Quantitative Chemical Estimation IRON ION: Fresh leaves of Justicia carnea were collected air dried and blended. 1g of the dried blended sample was taken and about 10mls of HCl was added and then heated to digest. Thereafter, this was allowed to cool and made up to 50mls volume, then read on atomic absorption spectrometry (A.A.S.) at a wave length of 248.3nm. POTASSIUM ION: Fresh leaves of Justicia carnea were collected air dried and blended. 1g of the dried blended sample was taken and about 10mls of HCl was added and then heated to digest. This was allowed to cool and made up to 50mls volume, then read on atomic absorption spectrometry (A.A.S.) at a wave length of 766nm. SODIUM ION: Fresh leaves of Justicia carnea were collected air dried and blended. 1g of the dried blended sample was taken and about 10mls of HCl was added and then heated to digest. Thereafter, this was allowed to cool and made up to 50mls volume, then read on atomic absorption spectrometry (A.A.S.) at a wave length of 589nm. RESULTS The Geographic location The geographic location of the parent is Port Harcourt, and Rivers State, Nigeria.

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Macro-morphological Study The macro-morphological description is shown in table 1. Justicia carnea JUST. See plate 1a, 1b and 1c.

Plate 1a: Justacia carnea Just., 1b.Flower inflorescence and 1c. Abaxial and adaxial foliar surfaces.

The leaves are of dark green, up to 15 to 20cm in length and 5 to 7cm in width observed facing plumes of tubular flowers. Table 1: summary of morphological characteristics of Justicia carnea JUST. Characters Justicia carnea Habit Evergreen shrub Duration Annual to biannual Root Tap Root system Stem Description Woody stem that grows up to 12 to 15m in height Leaf type Petiolate simple leaves Leaf organization Simple Phyllotaxy oppositely arranged Leaf outline or shape Ovate lanceolate Leaf margin Even or smooth Length of leaf (cm) 15cm Range 15 to 20cm Mean 18±3cm Breadth of leaf (cm) 5cm Range 5 to 7cm Mean 6±1cm Flower description plumes of tubular flowers with reddish corolla Fruit description

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Micro-morphological studies The foliar epidermal studies showed diacytic stomata and multicellular uniseriate trichomes which are amphistomatic. The foliar adaxial index of 34.1% and 28.2% for the abaxial. Plates 2a, and 2b.

Plate 2a: Justicia carnea Abaxial foliar Epidermis, 2b: J. carnea Adaxial foliar epidermis. White arrows in 2a and 2b indicate multicellular trichomes which are more in the adaxial regions whereas stomata are more numerous in the abaxial surface. ‘S’ represents stomata in both 2a and 2b. Anatomical Studies Anatomical study showed a layer of epidermal cells. The hypodermis is made of 5 to 14 rolls of collenchyma, general cortex and pith dominated by parenchyma in the same pattern of occurrence in mid-ribs, petioles, stems, nodes and roots except that the number of rolls of cells varied slightly and vasculation is open type.

Plate2: Justicia carnea Just. Petiole anatomy. 2a: Transverse Section (T.S.) of Petiole Revealing presence of glittering cystoliths indicated by cycle. 2b: Enlarged portion of the region of 2a cycled as indicated by arrow, showing a clearer view of the irregular shaped compound.

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Plate 3: J. carnea Just. Mid-rib (T.S.). Arrow revealed the pith having larger parenchyma at center while smaller ones nearer the vascular bundles. ‘H’ represents Hypodermis, ‘Gc’ stands for General cortex while ‘Vb’ is the vascular bundle. The qualitative phytochemical investigations conducted revealed presence of the following: alkaloids, flavonoids, tannins, anthraquinones, triterpenoids, cardenolide and saponins while phlobatannins and fixed oil components were absent. These are shown in table 2. Table 2: Qualitative Phytochemical Studies on Justicia carnea JUST. Phychemicals tested in F. exasperate Result Saponins +ve Alkaloids +ve Tannins +ve Phlobatannins -ve Anthraquinones +ve Flavonoids +ve Cardenolide +ve Steroids +ve Terpenoids +ve Fixed oil -ve Key: ‘+ve’ revealed ‘presence’ while ‘–ve’ showed ‘absence’ Quantitative Chemical Estimations The quantitative chemical estimation studied showcased 4200.1mg/kg of Sodium (Na), 15230.1mg/kg of Potassium (K) and 943.5mg/kg of Iron (Fe).The investigations conducted here could further be used to delimit the species. Table 3: Quantitative Chemical Estimations on Justicia carnea JUST. Specimen Iron (Fe) Potassium (K) Sodium (Na) 943.5mg/kg 15,230.1mg/kg 4200.1mg/kg

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DISCUSSION Occurrence of cystoliths in the vegetative parts in the petiole anatomy of Justicia carnea conformed to the findings of O’Neil (2010). Justicia carnea has simple trichomes elongated multicellular and glandular capitate trichomes and paracytic stomata which is in line with work of Amirul-Aiman et al. (2013). It is used as blood tonic, the vegetative parts turns deep red after boiling and could be administered for curative remedies due to the presence of relevant phytochemical constituents. CONCLUSION There is very limited number of research in micro-morphology of Justicia carnea. The proximate analysis, the karyotypes, quantitative aspect of phytochemistry and DNA barcodes may be of future interest.

ACKNOWLEDGEMENT The effort of Miss Kooro Justina B. who did the initial field collection of Justicia carnea and assisted in some of the laboratory research is immensely commended. REFERENCES 1. Borg, A.J. (2008). ‘Phylogenetics and Flora structure in Thumbergioideae and Avicennia (Acanthaceae) in Licentiate Thesis in Systematic Botany. Stockholm University. 2. Daniel, T. F. (2004). A Synopsis of Justicia Section Mesoamericanae (Acanthaceae) in Proceedings of the California Academy of Science. Pp. 174-183. 3. Reichert, E. T. (1913). Differentiation and Specificity of starch as in relation to genera, species etc. Carnege Inst. Washington Publ., 173. 4. Takeoka, T. (1962). Starch grains of endosperm in grass Systematics. Bot. Meg. Tokyo 75:277-383. 5. Brunswick, H. (1920). Uber das vorkommen Von Griphkriatallen der Tamericanceae. Sber Acad. Wiss. Wien (Math. Naturw. KI) 129:115-136. 6. Tomlinson, P. B. (1961). Anatomy of the monocotyledons-11. Palmae. Clarendon Press, Oxford. 7. Gulliver, G. (1966). On raphides as natural characters in the British flora. Quart. J. Micr. Sci. (N. S.) 6:1. 8. Gibbs, R. S. (1963). History of Chemical . In: Chemical Plant Taxonomy (Ed. T. Swain), Academic Press, London.

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