Taxonomic Characteristics and Phytochemical Constituents of Asystasia Gangetica (L.) T

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Asian Journal of Biology

11(4): 18-24, 2021; Article no.AJOB.67521 ISSN: 2456-7124

Taxonomic Characteristics and Phytochemical Constituents of Asystasia gangetica (L.) T. Anderson, a Member of Acanthaceae

C. Wahua1* and B. A. Odogwu1

1Department of Plant Science and Biotechnology, University of Port Harcourt, Choba, P.M.B. 5323, Nigeria.

Authors’ contributions

This work was carried out in collaboration between both authors. Author CW designed the study, performed the statistical analysis, wrote the protocol, wrote the first draft of the manuscript and managed the literature searches. Author BAO managed the analyses of the study. All authors read and approved the final manuscript.

Article Information

DOI: 10.9734/AJOB/2021/v11i430149 Editor(s): (1) Dr. Angelo Mark P Walag, University of Science and Technology of Southern Philippines, Philippines. Reviewers: (1) Ana-Alexandra Sorescu, The National Research & Development Institute for Chemistry and Petrochemistry, Romania. (2) Suica-Bunghez Raluca, The National Institute for Research & Development in Chemistry and Petrochemistry (INCDCP)- ICECHIM, Romania. (3) Ana Raquel Carneiro Ribeiro, Secretaria de Educação e Cultura do Estado do Rio Grande do Norte (SEEC-RN), Brazil. Complete Peer review History: http://www.sdiarticle4.com/review-history/67521

Received 14 February 2021 Original Research Article Accepted 24 April 2021 Published 29 April 2021

ABSTRACT

This study examined the taxonomic characteristics of Asystasia gangetica; a perennial herb characterized by its trailing to erect habit and as a result, roots from the nodes. It is glabrous and grows up to 80±20 cm. There are simple leaves with petioles which are ovate in shape with opposite phyllotaxy and margins are even to slightly serrated, measuring up to 7±2 cm in length and 3±1 cm in width. The inflorescence is one-sided raceme with purplish-blue tubular flowers which is pentamerous. The fruits are dry dehiscent capsule up to 2±0.5 cm in length. The epidermal studies revealed presence of simple elongated to conical shaped multicellular trichomes and cyclocytic stomata which are amphistomatic in nature, though very scanty in adaxial foliar region. Anatomical study showcased a single layer in the epidermis. The hypodermis consists of 2 to 3 layers of collenchyma, the general cortex and pith is made of parenchyma and cuts across mid- ribs, petioles, stems, nodes and roots, occurring in similar locations except that the number of

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*Corresponding author: E-mail: [email protected];

Wahua and Odogwu; AJOB, 11(4): 18-24, 2021; Article no.AJOB.67521

layers of cells are not the same and vascular bundle is open type. The phytochemical study revealed that the following secondary metabolites are present: alkaloids, saponins and cardiac glycosides while steroidal aglycones, not observed. This information here would assist for further delimitation of the species.

Keywords: Morphology; anatomy; phytochemistry; Asystasia; Acanthaceae.

1. INTRODUCTION together to get juice for the treatment of rheumatism [13]. People of Marudhamalai hills, Asystasia gangetica (L.) T. Anderson, is of the Coimbatore Tamil Nadu generally make use of family Acanthaceae, and is native to Asia and root paste for skin allergies [14], while in occurs throughout tropical Africa [1]. It is Kwazulu-Natal part of South Africa, it is used as commonly known as Chinese violet, Ganges vegetable. Some other ethnic groups the plant primrose, and coromandel synonymous to juice for anthelmintic activity, swollen areas and Justicia gangetica L. [2]. It has different rheumatism, gonorrhea and ear diseases [15]. It vernacular names such as Isihobo (Zulu) in also accounted in folk remedy for diabetes South Africa [1], in Nigeria it is referred to as mellitus in some regions of South India. In lobiri (Yoruba), Eboghogíro (Edo), Ekere Agukwu Nigeria, the leaves of Asystasia gangetica are (Igbo) [3]. It is a local traditional vegetable not highly effective in local treatment of asthma [16]. requiring cultivation and locally used as a It is no doubt that the therapeutic properties is potherb and leafy vegetable, mainly in times of due to the presence of certain biochemical such scarcity of other vegetable [4]. The plant has as carbohydrates, proteins, alkaloids, tannins, many medicinal, nutritional and local values. steroidal aglycones, saponins, flavonoids, and For instance, the leaves served as vegetable triterpenoids [17], the plant also contain minerals in some parts of Africa such as Kenya and like calcium, phosphorus, sodium, manganese, Uganda [4], and in Nigeria, it is used in trado- copper, zinc, magnesium, iron [18]. medicine for the treatment of asthma and sometimes used for embellishment in the Despite the economic and health benefits of environment [3]. Asystasia gangetica, the plant is neglected in terms research and conservation in Nigeria. The plant is an annual perennial, spreading or There is little information on the taxonomy and ground-reaching herb growing up to 60cm in phytochemical constituents of the plant in the height, with green, oval-shaped leaves arranged country (3, 4, 6, and 9). Hence, the relevance of in the opposite form, decurrently and broadly this article is to add more phenetic data to the triangular with quadrangular erect to semi existing knowledge of Asystasia gangetica (L.) T. straggling stem with white-cream coloured flower ANDERSON using both morphological and with purple markings and the fruit is a club phytochemical lines of evidence. shaped capsule, splitting from tip to base [5,6]. The micro-morphology showed cyclocytic type of 2. MATERALS AND METHODS stomata and presence of trichomes [7]. Peltate glandular trichomes and simple elongated 2.1 Geographic Location of Parent Plant multicellular-echinate ornamentation were present in Asystasia gangetica subsp. micrantha [8]. The presence of cymose or racemose The plant material used in this study was inflorescence and elastic loculicidal dehiscent collected fresh from the Centre for Ecological Studies, University of Port Harcourt, Rivers capsule with superior ovary is a strong property 0 1 11 0 1 11 of the Acanthaceae as a family [9]. The leaves (4 52 44 North, 6 55 20 East). A Tropical and floral parts are used as intestinal astringent. Rainforest zone of the Niger Delta region. It was One tablespoon of leaf juice mixed with equal identified at the University of Port Harcourt quantity of milk given in the morning and evening Herbarium domicile at the Department of Plant in empty stomach for diabetes [10,11]. It has Science and Biotechnology. been known from ancient time for its therapeutic values in Babungo for treating different diseases 2.2 Morphological Studies [12]. People who dwell in Sivagangai district of Tamil Nadu, peoples of Southern part of India For the morphological description, visual used the both the shoot and root systems observation of all plant parts was done and

Wahua and Odogwu; AJOB, 11(4): 18-24, 2021; Article no.AJOB.67521

compared with the aid of meter rule [5]. The having 2.411 LCD monitor and High sensitivity meter ruler was used for measurement involving ISO 1250. plant height from the root-collar to the terminal bud, the leaf length from the leaf tip to the 2.5 Phytochemical Study petiole base and the leaf width across the leaf The leaves of the specimen were sun dried for lamina, from one margin to another at the widest 72 hours and later weighed. Fifty grammes (50g) region. of the dried leaves were macerated in 96%

2.3 Epidermal Studies ethanol with a pestle and a mortar. The extract was filtered and then evaporated to dryness using a rotary evaporator set at 450 C. Fresh leaves collected in the morning (8 a.m.) for Residue yields were noted and a portion used for the purpose of reducing starch accumulation, the phytochemical investigation. were subjected to manual peeling and thereafter treated with alcohol solutions in the ratio of 50 %, 2.5.1 Test for alkaloids 75 % and absolute alcohol respectively. The cleared epidermis obtained were stained with This involved using 0.5 g of the plant extract, safranin for 5 minutes, rinsed with distilled water stirred with 5 mls of 1 % aqueous hydrochloric and counter stained with Alcian blue for 5 acid on a water bath; 1 ml of the filtrate was minutes, rinsed again and mounted in aqueous treated with few drops of Mayer’s reagent and a glycerol solution placed on glass slide with second 1 ml portion was treated in same way coverslip. Photomicrographs were taken from with Dragendorff’s reagent. The third 1 ml was good preparations. The stomatal index (S.I.) was treated with Wagner’s reagent as described by calculated using the formula: [20 and 21] and a modified thin-layer chromatography (TLC) method as described by [22] was used. A positive reaction on the S 100 S. I. = X chromatograms (indicated by an orange or S + E 1 darker colored spot against a pale yellow background) was used as confirmatory evidence Where S and E are mean numbers of stomata for the presence of alkaloid. and epidermal cells. Likewise the trichome Index 2.5.2 Test for flavonoids (T.I) was calculated using the formula: Shinoda reduction test: 5 g of the pulverized

T 100 sample was boiled in 5 mls of distilled water for 5 T. I. = X minutes on water bath and filtered while hot. T + E 1 Magnesium (Mg) was added to the filtrate and few drops of conc.H2SO4 were carefully Where T and E are trichomes and epidermal introduced into the mixture. The formation of cells, following the methods of [19]. orange, red, crimson or magenta was taken as evidence of preliminary presence of flavonoid. 2.4 Anatomical Studies Lead acetate test: 5 g of pulverized sample was Asystasia gangetica stems, leaves, petioles, boiled in 5 mls of distilled water for 5 minutes in flowers, fruits and roots harvested for the study, water bath and filtered while hot. 2 mls of 10 % were dehydrated in alcohol solutions of 50 %, 75 lead acetate was added to the filtrate and %, absolute alcohol and then passed through observed. Yellow precipitate indicated presence alcohol chloroform series in the ratio of 3:1 of of flavonoids. alcohol chloroform series, 1:1, 1:3 and pure 2.5.3 Test for tannins chloroform respectively for 5 minutes in each, and then rehydrated following same procedure to Ferric chloride test (FeCl3): 50 % alcohol before staining with safranin for 5 minutes and counter stained with Alcian blue 5 5 g of the prepared sample was boiled in 5 mls of minutes after rinsing. Free hand section was distilled water for 5 minutes on water bath. This done using the method described by [8]. was filtered while hot. 1 ml of 5 % FeCl3 was Microphotographs observed using the Light added to the filtrate and observed. Blue-black, compound microscope were done from good green or blue-green precipitate was taken as slides using Sony camera of 7.2 Mega pixels tannins present in the sample [21].

Wahua and Odogwu; AJOB, 11(4): 18-24, 2021; Article no.AJOB.67521

2.5.4 Test for anthraquinones 3. RESULTS

Borntrager’s test: Five grammes (5g) of each 3.1 Morphological Study plant extract were shaken with 10 mls benzene, filtered and 5 mls of 10 % ammonia solution The results for the morphological studies are added to the filtrate. The mixture was shaken presented in (Plates 1a to 1d and Table 1). From and the presence of a pink, red, or violet color in the result, it was observed that Asystasia the ammonia (lower) phase indicated that free gangetica is a perennial herb having trailing to hydroxyanthraquinones were present. erect habit with roots produced from the nodes. The stem is glabrous and grows up to 80±20 cm 2.5.5 Test for combined anthraquinones in height. There are simple leaves with petioles which are ovate in shape with opposite Five grammes (5g) of each plant extract was phyllotaxy and margins are even to slightly boiled with 10 mls aqueous sulphuric acid and serrated, measuring up to 7±2 cm in length and filtered hot and adhered to as described by [21]. 3±1 cm in width. The inflorescence is one-sided raceme with purplish-blue tubular flowers which 2.5.6 Test for phlobatannins is pentamerous. The fruits are dry dehiscent capsule up to 2±0.5 cm in length. The deposition of a red precipitate when an aqueous extract of the plant part was boiled with 3.2 Epidermal Study 1 % aqueous hydrochloric acid was accepted as evidence that phlobatannins were present in the The result of the epidermal study of the surface sample [21]. of the leaf is shown in (Plate 2). It was observed that the stomata are cyclocytic which is 2.5.7 Test for cardiac glycosides amphistomatic. Also, the lower epidermis has more stomata than the upper one. It was Lieberman’s test: 0.5 g of the extract was observed that trichomes were present. The dissolved in 2 mls of acetic anhydride and cooled trichomes were peltate and glandular trichomes in ice. One milliliter (1ml) of Sulphuric acid was which were in a simple, elongated and added in drops until a color change from violet to multicellular-echinate ornamentation. blue to green indicating that steroidal aglycones were present in the extract [23]. 3.3 Anatomical Study

2.5.8 Test for saponins The results of the anatomical studies of the stem, leaf node, leaf mid-rib, petiole and root were presented in (Plates 3a to 3f). From the result it Frothing tests preliminary following the method was observed that there were numerous vascular described by [24] was observed. Complete bundles in stem, mid-ribs and root. Although haemolysis of red blood cells around the disc there was absence of pith in the stem, the root after about 6 hours was taken as further had large central pith. evidence that saponins presence in sample.

Plate 1. Asystasia gangetica (L.) T. Anderson. 1a: The shoot and root system; 1b: The Inflorescence made of one-sided raceme and arrow indicates flower; 1c: The pentamerous purplish-blue tubular flower and 1d: Broadly spread tap root system

Wahua and Odogwu; AJOB, 11(4): 18-24, 2021; Article no.AJOB.67521

S S S

S 2a 2b 2c 2d

Plate 2. Asystasia gangetica adaxial and abaxial foliar epidermis. 2a, 2b and 2c: Adaxial

regions showing diverse structures. 2d: Abaxial surface revealing stoma and trichome,

very scanty in the upper regions. Arrow in 2a showcased peltate glandular trichome while 2b and 2c arrow revealed simple elongated and conical-shaped multicellular trichomes. ‘S’ is stomata in 2a, 2b, 2c and 2d respectively

h o Pt Pt Co Co 3c 3a 2 mm 2 mm 3b

T Pt Vb Xr Rb Hy Co 3d 2 mm 3e 3f 2 mm 2 mm

Plate 3. Asystasia gangetica anatomy. 3a: Stem section, 3b: Magnified portion of the stem anatomy in 3a showing the pith and other cell components clearer, 3c: Nodal section, 3d: Mid-rib anatomy, 3e: Petiole revealing 3 vascular traces and a pair of rib bundle wings; 3f:

Root anatomy. Keys: Vc is the vascular bundle, Pt – Pith, Hy – Hypodermis, Rb- Rib bundle wing, X - Xylary rays and vessel elements r Table 1. Summary of morphological characteristics of Asystasia gangetica

Characters Asystasia gangetica Habit Herbaceous Duration Annual perennial Root Roots are produced from the nodes Stem Description Trailing to erect and glabrous stem that grows up to 80±20 cm in height Leaf type Simple with petiole and cuneate at base Leaf venation type Reticulate Phyllotaxy Oppositely arranged Leaf outline or shape Ovate Leaf margin Entire to slightly serrated Length of leaf (cm) 7 cm Range 7.0 -9.0 cm Mean 7±2 cm Breadth of leaf (cm) Range 2.0 -4.0 Mean 3±1 cm Stipules Flower description Purplish-blue, tubular and pentamerous Fruit description A dehiscent capsule

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Table 2. Qualitative phytochemical studies on Asystasia gangetica (L.) T. Anderson

Extracts used for A. Alkaloids Saponins Cardiac glycosides Steroidal aglycones gangetica Water extract + ++ - - Methanol extract ++ + + - Ethanol extract +++ ++ + - Key: ‘+’ stands for presence ‘++’ denotes highly present ‘+++’ represents abundantly present

3.4 Phytochemical Study 5. CONCLUSION

The results of the phytochemical studies are Asystasia gangetica is a common weed in presented in (Table 2). From the results it was Nigeria. It is eaten as vegetable in some ethnic observed that alkaloids, saponins, cardiac groups and used for therapeutic administration in glycosides were present in the leaves of alleviating certain ailment, be that as it may, Asystasia gangetica but at different levels of more research investigations on the plant are concentrations. The phytochemicals, alkaloids needed because of the a foreseen potential very deeply present, when extracted with ethanol economic uses. Other areas necessitating than other extraction methods used, while research findings are: the quantitative aspect of steroidal aglycones were absent. phytochemistry, DNA barcodes and proximate analysis. 4. DISCUSSION ACKNOWLEDGEMENT The taxonomic lines of evidence showed that Asystasia gangetica belongs to the genus The effort of Mr. Amadi, Chinenmerem G. who Asystasia and the family Acanthaceae which is did the initial field collection of Asystasia supported by [1 and 8] though that is not in gangetica (L.) T. Anderson and assisted in some contention. of the laboratory research is immensely commended. From the gross morphology, it was observed that the plant is a perennial herb that had a trailing to COMPETING INTERESTS erect and glabrous stem, which produced roots from the node. The leaves are opposite, simple Authors have declared that no competing and ovate. This description conformed to those interests exist. of [2]. In the abaxial foliar epidermis, the stomata were cyclocytic and trichomes were observed. REFERENCES These findings are supported by the report of [3]. Stomata are considered to be one of the major 1. Lithudzha E. Asystasia gangetica (L.) structures within the leaf organ that have allowed Anderson subsp. micrantha (Nees) the higher plants to adapt to virtually all terrestrial Ensermu Pretoria NBG (June 2004) environments on the planet, by means of (additions by Yvonne Reynolds) S A adjustment of their size, density and distribution. National Biodiversity Institute; 2004. The presence of trichomes in Asystasia Plantzafrica.com gangetica, the peltate glandular trichomes and 2. Elliot L. Asystasia gangetica (L.) T. simple elongated multicellular-echinate Anderson Sub, sp. micrantha (Nees) ornamentation conform to the report of [7] and Ensermu. South Africa, National [8]. This finding further confirmed that the Biodiversity Institute. 2004; 2:34-50. Asytasia sp. used in this study was Asystasia 3. Akah PA, Ezike AC, Nwafor SV, Okoli CO gangetica. and Enwerem NM. Evaluation of the anti- asthmatic property of Asystasia gangetica The following are proven present in Asystasia leaf extracts. Journal of Ethno gangetica: alkaloids, saponins and cardiac Pharmacology. 2003; 89:25-36. glycosides as also observed with the work of [17] 4. Olufunke MD. Essential Oils from Aerial, though steroidal aglycones were observed Seed and Root of Nigerian Asystasia absent.

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