Journal of Ethnopharmacology 314 (2023) 116632 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm Pharmacognostic profiles, evaluation of analgesic, anti-inflammatory and anticonvulsant activities of Newbouldia laevis (P. Beauv.) Seem. ex Bureau leaf and root extracts in Wistar rats Cletus Anes Ukwubile a,*, Emmanuel Oise Ikpefan b, Musa Yusuf Dibal a, Vivian Amarachukwu Umeano c, David Nnamdi Menkiti d, Clement Chidi Kaosi e, Simon Paul f, Ademola Clement Famurewa g, Henry Nettey h, Timothy Samuel Yerima i a Department of Pharmacognosy, Faculty of Pharmacy, University of Maiduguri, Maiduguri, Nigeria b Department of Pharmacognosy and Traditional Medicine, Faculty of Pharmacy, Delta State University, Abraka, Nigeria c Department of Human Anatomy, Faculty of Medicine, University of Nigeria Nsukka, Enugu Campus, Enugu State, Nigeria d Department of Chemistry, Faculty of Physical Sciences, Ahmadu Bello University, Zaria, Nigeria e Department of Physiology, Faculty of Medicine, Chukwuemeka Odumegwu Ojukwu University, Nigeria f Department of Biological Sciences, Faculty of Science, Federal University of Health Sciences, Otukpo, Nigeria g Department of Medical Biochemistry, Faculty of Basic Medical Sciences, College of Medicine, Alex Ekwueme Federal University, Ndufu-Alike Ikwo, Nigeria h Department of Pharmaceutics and Pharmaceutical Microbiology, School of Pharmacy, University of Ghana, Legon, Ghana i Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Maiduguri, Maiduguri, Nigeria A R T I C L E I N F O A B S T R A C T Keywords: Ethnopharmacological relevance: Newbouldia laevis is a popular medicinal plant whose leaves and roots are used Pharmacognostic profiles in Nigeria as ethnomedicinal prescriptions for pain, inflammation, convulsion, and epilepsy. These claims have Newbouldia laevis not been scientifically verified prior to this study. Analgesic Aim of the study: To determine pharmacognostic profiles of the leaves and roots and evaluate the analgesic, anti- Anti-inflammatory Anticonvulsant inflammatory, and anticonvulsant activities of methanol leaf and root extracts in Wistar rats. Material and methods: The pharmacognostic profiles of the leaves and roots were determined using standard procedures to serve as fingerprints for the plant. The methanol leaf and root extracts of Newbouldia laevis were tested for acute toxicity using the OECD’s up and down method at the maximum dose of 2000 mg/kg (orally) in Wistar rats. Analgesic studies were carried out in acetic acid-induced writhing in rats and tail immersion. The anti-inflammatory activity of the extracts was evaluated using carrageenan-induced rat paw-oedema and formalin-induced inflammation in rats’ mode. The anticonvulsant activity was determined using strychnine- induced, pentylenetetrazol-induced, and maximal electroshock-induced rat convulsion models. For each of these studies, the extracts doses of 100, 200 and 400 mg/kg were administered to the rats following the oral route. Results: The pharmacognostic profiles showed that the leaves possessed deep-sunken paracytic stomata (5-8-16 mm2; adaxial, 8-11-24 mm2; abaxial epidermis), vein islets (2-4-10 mm2; adaxial), vein terminations (10-14-18 mm2; adaxial), palisade ratio (8.3-12.5-16.4 mm2; adaxial, 2.5-6.8-12.2 mm2; adaxial), covering unicellular trichome (8–14; adaxial), spheroidal calcium oxalate crystals (3–5 μm), and oval-shaped striated starch grain with no hilum (0.5–4.3 μm). The transverse section of the leaf showed the presence of spongy and palisade parenchyma as well as a closed vascular bundle. The root powder showed the presence of brachy sclereid, fibers without lumen, and lignin. All physicochemical parameters fall within the acceptable limits, phytochemical contents showed mainly glycosides, alkaloids, and steroids while acute oral toxicity (LD50) of the parts for 14 days did not produce any toxicity signs or mortality in the rats. The extracts produced dose-dependent (100–400 mg/kg) analgesic involving opioid receptors, anti-inflammatory, and anticonvulsant activities in the rats which were significant (p ≤ 0.05) when compared to the standard drugs. The leaf extract possessed the most potent analgesic and anti-inflammatory effects in the rats, while the most anticonvulsant effects were observed in rats * Corresponding author. Department of Pharmacognosy, Faculty of Pharmacy, University of Maiduguri, Bama Road Maiduguri, Borno State, Nigeria. E-mail addresses: doccletus@yahoo.com, caukwubile@unimaid.edu.ng (C.A. Ukwubile). https://doi.org/10.1016/j.jep.2023.116632 Received 9 April 2023; Received in revised form 9 May 2023; Accepted 10 May 2023 Available online 19 May 2023 0378-8741/© 2023 Elsevier B.V. All rights reserved. C.A. Ukwubile et al. J o u r n a l o f E t h n o p h a r m a c o l o g y 314 (2023) 116632 treated with the leaf extract. Both extracts showed elevated levels of protection against strychnine-induced, pentylenetetrazol-induced, and maximal electroshock-induced seizure in rats. Conclusion: Our study revealed some pharmacognostic profiles of Newbouldia laevis leaves and roots that are vital for its identification from closely related species often used for adulteration in traditional medicine. The study further showed that the leaf and root extracts of the plant possessed dose-dependent analgesics, anti- inflammatory and anti-convulsant activities in rats, thus, justifying its use for the treatment of these diseases in Nigerian traditional medicine. There is a need to further study its mechanisms of action towards drug discovery. the medicinal plants’ products (Lalthanpuii and Lalchhandama, 2020; Ukwubile et al., 2019; Zhang et al., 2019). Presently, several important orthodox medicines such as artemisinin, Abbreviations vincristine, vinblastine, quinine, morphine, galanthamine, atropine, among others were derived from bioactive compounds isolated from LD50 Median lethal dose medicinal plants for the treatment of vasrious diseases pains, in- NSAIDs Non-steroidal anti-inflammatory drugs flammations, convulsions, cancer, malaria, etc. (Alamgir, 2017; Jones SEM Scanning electron microscope et al., 2006; Mohammed Golam Rasul., 2018; Nonglang et al., 2022; TLC Thin layer chromatography Prior et al., 2005). For instance, ailment such as pain is a sign in within GC Gas chromatography the CNS indicating certain abnormality in the body. It is often charac- MSD Mass spectrum detector terized by symptoms like ache, tingle, burn, or prick which may be NMR Nuclear magnetic resonance short-lived or lasting. In most cases, pain can be a sign for underlying FTIR Fourier transform infra-red disease or certain pathological condition of an organ (Pandey et al., CNS Central nervous system 2020). Currently, treatment of pains is done using various types of an- GABA Gamma-aminobutyric acid algesics or NSAIDs with some levels of adverse effects in the body. i.p. Intraperitoneal Because of these undesirable side effects resulting from the intake of b.w. Body weight these pain-killers, research into the treatment of pains (acute or chronic) ANOVA Analysis of variance using medicinal plants have gained much attention due to the numerous SPSS Statistical package for social sciences advantages of drugs from plants over conventional medicines (Alam et al., 2020). The use of plants to treat pains have successfully yielded the desired results especially on chronic pains experienced by older adults (Sharma et al., 2020). 1. Introduction Similarly, inflammation is an immunological response by the body due to invasion of microorganisms like viruses and bacteria often Medical plants are now been to considered as important sources for resulting in swelling, redness, heat, and pain in the affected part of the managing, treating and preventing different types of diseases (Yadav body. It may be acute or chronic inflammation condition which is Abhishek and Samanta Krishanu, 2021). All plants contain important associated with diseases like asthma, cancer, diabetes, Alzheimer’s phytochemicals that can be used as drug agents that act as lead com- disease, and heart problems (Lee et al., 2019; Nainwani et al., n.d.). pounds in the development of various types of orthodox medicines Treatment of inflammations is usually done using NSAIDs which have (Jones et al., 2006). Nowadays, many less developed or developed been reported to have caused several side effects in the body. Many plant countries of the world are patronizing medicinal plants or their products extracts have been successfully used in traditional medicine for treating for some important aspects of human healthcare such as treatment of inflammations, examples include Vernonia puaciflora, Bidens pilosa, diseases like malaria, cancer, inflammation, pains, ulcer, diabetes, in- Spondias venulosa, etc., (U. C. Anes, 2015; Mohammed Golam Rasul., fections, etc. These medicinal plants include garlic, gingko, aloe, grav- 2018; Pandey et al., 2020). iola, ginseng and Catharanthus, among others (Yadav Abhishek and On the other hand, disease such as convulsion occurs because of Samanta Krishanu, 2021). Ethnomedicinal uses of these medicinal involuntary contraction of muscles especially in children, although it plants for decades have shown some prospects in future due to wide occurs also in adults. It is very common in epileptic seizure and can also distribution of over 5000 species of plants throughout the world occur due to high fevers, brain traumas and infections (Dighe and Barve, occurring in both terrestrial and aquatic habitats. Most of these plants 2019). Convulsion can be localized in a particular part of the body or the have not been evaluated ethnopharmacogically and their biological whole body resulting in seizures, though not all seizures cause convul- activities could be useful in the treatment of some difficult or incurable sion (Akhigbemen et al., 2019). Factors such as sudden rise in fever, diseases in the future (Yadav and Samanta, 2021). certain epileptic seizure, hypoglycemia, and tetanus are often tagged Currently, ethnomedicinal uses of plants have been improved upon symptoms of convulsion. Many plants have been researched upon to be by various research in an aspect of pharmacy termed pharmacognosy very effective in the treatment convulsion in various parts of the world. (Alamgir, 2017; Jones et al., 2006). Pharmacognosy has been defined as Some of these plants include thyme, cannabis, cloves, ginger, turmeric, the scientific study of drug of natural origin as well as their ethnobotany, tetrapleura, flowers of Newbouldia laevis and cowhage. These plants methods of collection, modes of preparation, standardization tech- many bioactive compounds that were potent of convulsion and epileptic niques, ethnopharmacology, cultivation, conservation, and commerce. seizures (Akhigbemen et al., 2019; Dighe and Barve, 2019; Olatokunboh Pharmacognosy as an aspect of natural science consist of various disci- et al., 2009; Shelar et al., 2018; Shinde et al., 2018; Yeddes et al., 2022). plines such as phytochemistry, ethnopharmacology, economic botany, The plant Newbouldia laevis is a rapid-growing tropical tree of 3–8 m toxicology, systematics, ethnobotany, taxonomy, microbiology, phar- high in western and about 20 m high in eastern and northern Nigeria maceutics, and biotechnology. Since about 80% of the rural population belonging to the Family Bignoniaceae. It commonly called African in the world depends of drugs from natural sources for their primary hyssop, ‘Ogirishi’’ in Igbo, ‘Àdùrúúkù’’ in Hausa, and ‘’Akoko’’ in Yoruba healthcare, pharmacognostic profiles of these drugs are of utmost (Nigeria). The plant is widely distributed in Nigeria, Senegal, Ghana, DR important for quality control and standardization of herbal medicines Congo, Sierra Leon, Benin republic and South American countries like (Jones et al., 2006). Evaluation of the pharmacognostic profiles of Brazil. All the parts of the plant have been used to treat various diseases herbal drugs will greatly help to ensure the efficacy, safety and quality of 2 C.A. Ukwubile et al. J o u r n a l o f E t h n o p h a r m a c o l o g y 314 (2023) 116632 in traditional medicine such as malaria (stembarks), diabetes, pile value, extractive values, etc. (stembarks), epilepsy (flowers), and juice (Ndidi et al., 2020). In Nigeria, the leaf, stembark, and root decoctions are used as analgesic, 2.3.3. Phytochemical analysis of extracts anti-inflammation and anticonvulsant or epileptic agent in traditional Phytochemical contents of methanol leaf and roots extracts were medicine (Iwu, 2014). Although, some of these claims on the uses have evaluated using the standard methods to test for the presence of certain not been verified scientifically, yet, the use of these plant parts as eth- metabolites (Bijauliya et al., 2021; Kumar et al., 2015; Olaleye et al., nomedicinal prescription for pain, inflammation (reducing swellings) 2021; Ukwubile, 2017). and convulsion (or epilepsy) have gained more acceptance in most rural communities in South east Nigeria (Iwu, 2014). The bole is cylindrical 2.3.4. Isolation and characterization of bioactive compounds from extracts up to 90 cm in diameter, often used for marking boundaries and fences The crude methanol leaf and root extracts were separately subjected in Nigeria, besides its ethnomedicinal uses. The juice from its flowers to silica gel column chromatography using gradient elution technique. A contain significant amount of sugar, and has become very attractive to total of twenty fractions were collected from each extract and grouped children who lick the flowers (Iwu, 2014; Pages et al., 2013). The leaves into four groups A to D based on their profiles on TLC plates (Gel 60 of the plant contain tannins, alkaloids, saponins, cardiac glycosides, F254 Merck, Chemtech Intl. Gujarat India). Fractions with most potent flavonoids and anthraquinones (Dermane et al., 2020). activity were purified using short column chromatography, purity was Therefore, this present study was carried out to determine some confirmed by single TLC spot (Eiceman et al., 1994; Kim et al., 1998). important pharmacognostic profiles of leaves and roots of Newbouldia Characterization of bioactive compounds was done using an Agilent laevis with a view to identifying possible adulteration from closely technologies GC (7890A) coupled to a MSD (5975C) with optimal related species, and evaluate the analgesic, anti-inflammatory and operating conditions (Alam et al., 2020). Compounds were compared anticonvulsant activities of methanol leaf and root extracts induced with those in the NIST library (Ukwubile, Ahmed, Katsayal, Ya’u et al., Wistar rat models. 2019). 2. Materials and methods 2.4. Experimental animals 2.1. Collection, identification, and authentication of plant material Adult Wistar rats of opposite sexes numbering one hundred and twenty (120) and weighing 100–150 g were purchased from PJ Rats Fresh leaves and roots of Newbouldia laevis was collected in early Farm Ltd, Jos, Nigeria. The animals were acclimatized in the laboratory morning hours from a forest in Nsukka, Enugu State, Nigeria. The at 25 ± 4 ◦C, 10% humidity, 12 h light and dark light cycles for 7 days identification and authentication of the plant was done at the herbarium with free access to water and food. Approval for the use of the was ob- unit of the Department of Pharmacognosy, Faculty of Pharmacy, Uni- tained from the Animal in research ethical group of PJ Rats Farm Ltd versity of Maiduguri, Nigeria, where a voucher specimen number UMM/ with approval number: PJF/NG/JOS-035-2023. FPH/BIN/001 was deposited for the plant at the herbarium unit. 2.2. Preparation of plant extracts 2.5. Acute oral toxicity study The collected fresh leaves and roots of N. laevis were carefully rinsed Five (5) Wistar rats of both sexes weighing between 100 and 120 g in water to remove unwanted debris and dirt. They were shade-dried for were administered maximum dose 2000 mg/kg extracts and monitored two weeks in free air. The shade-dried plant materials were then ground for signs of toxicity especially within the first 4 h. The rats were there- into fine powders using electrical blender (model: BLG 1500 PRO, after observed for 14 days for behavioral changes like redness of eyes, Binatone, Nigeria) and weighed. Powdered samples of leaves and roots itching or mortality (Khalifa, 2022). weighing 1000 g each were extracted with 100% methanol (Sigma Aldrich, St Louis, Mo, US) using cold maceration technique for 72 h. The 2.6. Analgesic activities of extracts extract was each evaporated to dryness in a rotary evaporator (Buch, UK) to obtain greenish and brownish jelly-like extracts of leaves (16.4% 2.6.1. Acetic acid-induced writhing in rats w/w yield) and roots (8.4% w/w yield) respectively. The extracts were The analgesic activity of N. laevis leaf (NLE) and root extracts (NRE) weighed and stored in a refrigerator at 10 ◦C for further use. were evaluated using acetic acid-induced writhing test in rats. Briefly, rats of opposite sexes weighing 100–150 g were grouped into five groups 2.3. Pharmacognostic studies on N. laevis leaf and root powders of rats per group. Group I (negative control) was administered 10 mL/kg distilled water, group II (positive control) was administered 100 mg/kg To standardize the leaves and roots of the plant, we carried out standard drug diclofenac sodium (Hovid Div. Phamatex Nig. Ltd), while various aspects of pharmacognostic studies to serve as profiles (finger- groups III, IV and V were administered 100, 200, and 400 mg/kg extract print) for proper identification of the plant. doses of leaf and root intraperitoneal (i.p.). After 30 min, 0.5% acetic acid (10 mL/kg b.w.) was injected into the rats (i.p.). Five minutes after, 2.3.1. Quantitative microscopy of leaves and roots the number of abdominal writhing was observed using magnifying Some important qualitative microscopic features of the leaf such as handheld lens for 30 min in triplicate. The percentage inhibition of stomatal number, stomatal type, stomatal index, vein islet number, vein writhing was calculated from the formula below (Alam et al., 2020). termination number, palisade ratio, starch grains, trichomes, calcium % inhibition = (Nc-Nt/Nc) 100 (i) oxalates, and transverse section as well as other profiles of leaves and roots were determined following previously described procedures Where, Nc represents mean number of writhing in control group, and Nt (Dodiya and Jain, 2017). The nature of stomata of the leaf was observed is mean number of writhing in treatment groups. using Phenom desktop SEM (ThermoFisher Scientific, MA, USA). 2.6.2. Tail immersion test in rats 2.3.2. Physicochemical evaluation of powdered leaves and roots The rats were grouped into five groups of five rats per group. Food The physicochemical parameter of leaves and roots were determined and water were withdrawn from the animals 2 h prior to the using the procedures previously described (C. Anes et al., 2023; Dodiya commencement of the experiment. Group I (negative control) received and Jain, 2017), to evaluate parameters such as moisture contents, ash 10 mL distilled water, group II (positive control) received 50 mg/kg 3 C.A. Ukwubile et al. J o u r n a l o f E t h n o p h a r m a c o l o g y 314 (2023) 116632 pentazocine (Biopharma Nig. Ltd), while groups III, IV and V received 2.8.3. Maximal electroshock-induced rat convulsion extracts dose of 100–400 mg/kg (i.p.). After 1 h, 3 cm of the tail in each Rats were randomly grouped into five of rats per group following rat was immersed in water bath (Isotemp GPD 10, Fisher Scientific) previously described protocol (Mante et al., 2013). Group I received 10 containing hot water (50.5 ◦C). The reaction time was taken as time mL/kg distilled water, group II received 20 mg/kg phenobarbital taken by each animal to withdraw or flick its tail from the hot water. A (Emzor Pharm. Industries Ltd, Nig.), groups III, IV and V received extract 20 s cut-off or no response time was noted, and reaction time was doses 100, 200, and 400 mg/kg (i.p). After 1 h, the rats were exposed to measured in 0, 15, 30, 45 and 60 min (Sharma et al., 2020). electroshock at 150 mA, 0.2s using a pair of ear clip electrodes (Ugo Basile). The commencement of tonic-hind limb extension and its pro- 2.6.3. Investigation of opioid receptors involvement in analgesic activity of tection was recorded (Dighe and Barve, 2019). extracts The involvement of opioid receptors in analgesic activity of N. laevis leaf and root extracts was determined using a nonselective opioid re- 2.9. Statistical analysis ceptor antagonist naloxone (Alpha Pharmacy & store Ltd). Briefly, Wistar rats weighing 100–130 g were randomly grouped into five groups Data were expressed as mean ± SD (n = 5). Significant difference of five rats. Group I received 10 mL/kg distilled water, group II received between the control and treatment groups were considered at p ≤ 0.05 2 mg/kg naloxone, group III received 50 mg/kg pentazocine, groups IV using one-way ANOVA followed by Dunnett’s post-hoc test. Statistical received 400 mg/kg extracts doses while groups V and VI received 400 analysis was done using SPSS statistical software version 22. mg/kg plus naloxone and 25 mg/kg pentazocine plus naloxone (i.p.). Fifteen (15 min) prior to administration of the samples to rats groups V 3. Results and VI, naloxone was given to the animals (Hijazi et al., 2017). 3.1. Evaluation of pharmacognostic parameters 2.7. Evaluation of anti-inflammatory activity of extracts 3.1.1. Quantitative microscopy and histological features of leaf and root Quantitative microscopic examination of N. laevis fresh leaf showed 2.7.1. Carrageenan-induced paw-oedema test it contained a paracytic type of stomata that are numerous on the lower To evaluate the anti-inflammatory activity of the extracts surface than the upper surface. Similarly, few numbers of glandular carrageenan-induced paw oedema rat model was used following previ- unicellular trichomes were observed on the upper surface. There are no ously described protocol (Pandey et al., 2020; Sharma et al., 2020). epidermal cells resembling each other in the leaf epidermis (Table 1; Briefly, twenty-five (25) Wistar rats grouped into five groups of five rats Fig. 1). per group. Group I received 10 mL/kg distilled water, group II received The transverse section (TS) of the leaf showed it contain two 100 mg/kg standard drug diclofenac sodium (Phamatex Nigeria Ltd), parenchymatous cells (palisade and spongy parenchyma), closed group III, IV and V were administered 100, 200 and 400 mg/kg extract vascular bundles without cambium, and moderately thick cuticle doses respectively. After 1 h, carrageenan (0.1% w/v dissolved in 0.1% (Fig. 2a). In the root powder, there are lignin substance, prismatic cal- normal saline) was given to the rats’ sub-plantar area of the paw in right cium oxalate crystals, fiber sclereids with narrow lumen, and concentric hind limb. The volume of paw oedema was measured at 0, 1, 3, and 5 h vascular bundles, while the TS showed a wide pith and closed vascular using plethysmometer (model: 37140, Ugo Basile). The percentage in- bundle (Fig. 2b). hibition of inflammation was then calculated from the formula shown below: 3.1.2. Physicochemical evaluations of leaf and root powders The study showed that moisture content for leaf powder was 6.52 ± % Inhibition of oedema = (PVc – PVt/PVc)100 (ii) 0.01% w/w and 8.04 ± 0.10% w/w for root powder. The ash value for Where, PVc denotes paw volume of control, and PVt denotes paw vol- the leaf powder was the least with value of 4.24 ± 0.01% w/w while the ume of treated. root had the highest value of 12.06 ± 1.02% w/w. Furthermore, water extractive value of the leaf powder was the highest with the value of 2.7.2. Formalin-induced inflammation in rats 10.28 ± 1.02% w/w while, the root powder had 6.20 ± 0.01% w/w. Twenty-five (25) rats were grouped as in carrageenan-induced paw From the results, other parameters fall within the acceptable ranges oedema test above except that trats were induced using formalin (Table 2). following the method previously described (Sharma et al., 2020). 3.1.3. Phytochemical contents of leaf and root extracts The phytochemical contents showed the presence of more metabo- 2.8. Evaluation of anticonvulsant activities of extracts lites in the leaf extract than the root. The results revealed the presence of tannins, flavonoids, alkaloids, triterpenes, saponins, and carbohydrates 2.8.1. Strychnine-induced rat convulsion in the leaf extract while the root extract indicates the presence of fla- Twenty-five Wistar rats of opposite sex were randomly grouped into vonoids, alkaloids, cardiac glycosides, triterpenes and saponins five groups of five. Group I received 10 mL/kg distilled water, group II 4 mg/kg diazepam (Michelle Labs. healthcare), while groups I III, IVand V Table 1 received extract doses 100, 200 and 400 mg/kg (i.p.). After 1 h, 2 mg/kg Leaf surface data of N. laevis as determined using the microscope (40x). strychnine (Bio-Techne Ltd, UK) was injected (i.p.) into the rats. The rats Leaf Parameter Quantity/mm2 were then observed for 30 min for the onset of convulsion or death Upper surface Lower surface (Mante et al., 2013). Stomatal number 8-6.2-14 12-10-22 Stomal index 12.14% 18.22% 2.8.2. Pentylenetetrazole-induced rat convulsion Vein terminations number 10-8.5-16 4-3.8-10 Twenty-five Wistar rats were grouped as previously described Vein islets number 6-5.1-12 5-3.4-8 (Mante et al., 2013). After 1 h of administering (i.p.) the test samples, 80 Palisade ratio 14.22 Na mg/kg pentylenetetrazole (Sigma-Aldrich, St Louis Mo, USA) was given Trichomes 5-4.5-7 Na to the animals. Observation for the onset of tonic-clonic convulsion or Numbers in bold are mean of repeated counts (n = 5), Na (not applicable). deaths of rats was made for 30 min period (Obese et al., 2021). Glandular unicellular trichomes were observed on the upper surface of leaf. 4 C.A. Ukwubile et al. J o u r n a l o f E t h n o p h a r m a c o l o g y 314 (2023) 116632 Fig. 1. Scanning electron microscopy (SEM) of N. laevis leaf (a) Upper surface (b) lower surface; 800x. St; stomata, Su; subsidiary cells. Fig. 2. The TS leaf and root of N. laevis showing various anatomical features. Ue; upper epidermis, Xy; xylem tissue, Ph; phloem tissue, Sp; spongy parenchyma, Pp; palisade parenchyma, Ct; cuticle, La; leaf lamina, Vb; vascular bundles, Co; collenchyma, Pa; parenchyma, Sc; sclerenchyma (with sclereid), Pt; pith, 40x. (Table 3). Table 2 3.1.4. GC-MS profiles of leaf and root extracts Physicochemical parameters of Newbouldia laevis leaf and root. The GC-MS analysis of the isolated bioactive compounds from the Parameter (% w/w) Value (mean ± SD) leaf and root extracts showed the presence of mainly cyclic and aromatic Leaf Root compounds as well as unsaturated fatty acid esters. Similarly, some alkaloidal compounds were also revealed in the leaf such as pyridin-3-yl- Moisture contents 6.52 ± 0.01 8.04 ± 1.10 Ash value 4.24 ± 1.01 12.06 ± 1.02 ethanimidamide (m/z: 135.11 g/mol) and 2-N-butylacrolein (m/ Total ash 8.58 ± 1.11 14.10 ± 1.04 z:112.17 g/mol), while in the root extract only one alkaloidal compound Water soluble ash 2.02 ± 0.01 2.88 ± 0.01 2-furanmethanamine (m/z: 97.12) was isolated (Tables 4 and 5). Alcohol insoluble ash 0.80 ± 0.01 1.64 ± 0.02 Water extractive 10.28 ± 1.02 6.20 ± 0.01 Alcohol extractive 4.01 0.01 8.44 1.04 3.1.5. Acute oral toxicity of leaf and root extracts ± ± Dry matter 12.40 ± 2.02 10.14 ± 1.02 The study showed that at maximum dose of 2000 mg/kg body weight (b.w.) administered to the animals (oral), there were no signs of toxicity Results are mean ± SD (n = 3). after two weeks of repeated administration of extracts. The LD50 was Table 4 Isolated compounds from N. laevis leaf extracts analyzed by the GC-MS. Table 3 Phytochemical contents of Newbouldia laevis leaf and root extract. Compounds Peak area Retention time m/z (g/ (%) (min) mol) Constituents Leaf Root 5-Methyl-1-heptanol 3.19 5.56 129.15 Tannins + – 2-Octanol-S-ester 4.98 8.99 130.02 Flavonoids + + Pyridin-3-yl- 7.66 12.18 135.11 Anthocyanins – – ethanimidamide Cyanogenic glycosides – – 2-Ethylformanilide 3.61 13.92 149.23 Alkaloids + + Benzonitrile, 2-fluoro 4.35 14.05 121.04 Cardiac glycosides – + N-Benzyl formamide 9.02 18.27 117.10 Triterpenes + + 1,3-Benzoxazol-4-ol 12.98 17.85 135.12 Saponins + + N-Tridecan-1-ol 11.27 20.96 200.36 Carbohydrates + – Melibiose 8.45 15.09 342.30 Note: + (detected), - (not detected). 2-N-butylacrolein 6.06 25.68 112.17 5 C.A. Ukwubile et al. J o u r n a l o f E t h n o p h a r m a c o l o g y 314 (2023) 116632 Table 5 12 Isolated compounds from N. laevis root extracts analyzed by the GC-MS. 10 Compounds Peak area Retention time m/z (g/ (%) (min) mol) 8 Benzoic acid, ethyl ester 55.53 5.22 150.10 6 Control 1-Hexene, 3,5-dimethyl 1.67 8.96 112.21 Standard 2-Octanol, (S)-ester 4.98 8.99 130.02 4 400 mg/kg LE 1,6-Octadiene, 3,7-dimethyl- 12.97 15.77 138.25 2 400 mg/kg RE(S) 2-Furanmethanamine 2.79 16.14 97.12 0 1,10-Decanediol 4.47 16.42 174.28 0 min 30 min 60 min 90 min 120 min Hydrazine, 1,2-dimethyl 4.44 17.83 60.09 -2 3-Aminocrotononitrile 2.26 20.38 82.10 Time (min) Fomepizole 11.49 34.94 82.11 2,6-Nonadienal, (E,E)- 1.86 15.06 138.21 (a) 10 Table 6 9 Acute oral toxicity of N. laevis methanol leaf and root extract in rats. 8 Group (mg/kg) Animal died/Animal survived 7 Standard Leaf extract Root extract 6 Nal + 400 mg/kg LE 5 I (2000) 0/1 0/1 Nal + 400 mg/kg RE II (2000) 0/1 0/1 4 400 mg/kg LE III (2000) 0/1 0/1 3 IV (2000) 0/1 0/1 400 mg/kg RE2 V (2000) 0/1 0/1 1 Nal + Penta LD50 > 2000 mg/kg b.w. 0 0 min 30 min 60 min 90 min 120 min Time (min) (b) 90 Fig. 4. Effects of N. laevis extracts on reaction time (a) and change in latency 80 time in induced rats. Results are mean ± SD (n = 5), 400 mg/kg LE: leaf extract, 70 400 mg/kg RE: root extract, Nal: naloxone, Penta: pentazocine. There was 60 0 min statistical significance difference when compared to control (p ≤ 0.05; one-way 50 10 min ANOVA followed by Dunnett’s post-hoc test). The standard drug used was pentazocine. 40 15 min 30 20 min estimated to be greater than 2000 mg/kg b.w. leaf and root extract 20 25 min (Table 6). 10 30 min 0 3.2. Evaluation of analgesic effects of extracts Control Standard 100 mg/kg 200 mg/kg 400 mg/kg Group 3.2.1. Acetic acid-induced writhing test The results from acetic acid-induced writhing in rats revealed that N. laevis extracts displayed dose-dependent reductions in number of abdominal writhing in rats. These decreases were witnessed more in the 100 leaf extract at 400 mg/kg dose in 30 min duration than the root extract. 90 These results were significance (p ≤ 0.05; one-way ANOVA) when 80 compared to the control groups (Fig. 3 a and b). 70 0 min 60 3.2.2. Effect of N. laevis extracts on rats’ tail flick responses 10 min From the results obtained, there was significant decrease in reaction 50 15 min time of responses by the animals after administration of 400 mg/kg dose 40 20 min of leaf and root extracts orally. These results were statistical significance 30 25 min (p ≤ 0.05) when compared to standard drug pentazocine (Fig. 4 a and b). 20 30 min 10 3.3. Evaluation of anti-inflammatory effects of N. laevis extracts 0 Control Standard 100 mg/kg 200 mg/kg 400 mg/kg 3.3.1. Carrageenan-induced rat paw oedema Group The results showed that carrageenan-induced paw oedema decrease paw diameter of rats in dose-dependent fashion. However, the leaf extract showed greater potency on paw oedema volume reduction than Fig. 3. Effects of N. laevis (a: leaf) and (b: root) extracts on abdominal writhing the root extract in both carrageenan and formalin-induced paw oedema in rats. Results are mean ± SD (n = 5), p ≤ 0.05 (one-way ANOVA followed by experiments (Fig. 5 a and b). These results were also signficant when Duncan’s multiple range test). compared to control (p ≤ 0.05). 6 Number of writhings (%) Number of writhing (%) Change in latency time (Sec) Reaction time (% response) C.A. Ukwubile et al. J o u r n a l o f E t h n o p h a r m a c o l o g y 314 (2023) 116632 1.4 Table 8 Anticonvulsant effects of N. laevis extracts on pentylenetetrazole (PTZ)-induced 1.2 seizure in rats. 1 Group dose Latency Recovery time Death/total (% (min) (min) mortality) 0.8 Control 10 mL distilled 0.82 ± 0.01 82.14 ± 4.22 4/5 (80; Standard water protection:20%) 0.6 400 mg/kg LE 2 mg/kg 44.11 ± 1.64 ± 0.02* 0/5 (0; protection: diazepam 2.14* 100%) 0.4 400 mg/kg RE 100 mg/kg LE 52.08 ± 8.02 ± 0.01 0/5 (0; 1.15 protection:100%) 0.2 200 mg/kg LE 60.02 ± 12.08 ± 1.01* 3/5 (60; protection 2.01* 40%) 0 400 mg/kg LE 25.22 ± 10.01 ± 0.01* 0/5 (0; 1 h 2 h 3 h 4 h 5 h 2.01* protection:100%) Time (hour) 100 mg/kg RE 33.08 ± 23.00 ± 2.01 0/5 (0; 2.02 protection:100%) (a) 200 mg/kg RE 38.64 ± 16.24 ± 1.01* 4/5 (80; 3.01* protection:20%) 400 mg/kg RE 30.12 ± 12.02 ± 1.01* 0/5 (0; 3.5 2.01* protection:100%) 3 Results are mean ± SD (n = 5),*statistical significance at p ≤ 0.05 (one-way ANOVA followed Dunnette’s post-hoc), LE: leaf extract, RE: root extract. 2.5 2 Control 3.4.2. Pentylenetetrazole (PTZ)-induced convulsion in rats Standard In Table 8 below, 100% protection of the rats was achieved when the 1.5 400 mg/kg LE animals were given low dose (100 mg/kg) and high dose (400 mg/kg) of 1 400 mg/kg RE extracts while the medium dose (200 mg/kg) does not offer 100% pro- tection on the animals. A similarly results was obtained when 2 mg/kg 0.5 diazepam (standard drug) was given to the animals (i.p.) where 100% protection was produced. 0 1 h 2 h 3 h 4 h 5 h Time (hour) 3.4.3. Maximum electroshock (MES)-induced convulsion in rats There was dose-dependent decrease in onset of convulsion and duration of convulsion in rats exposed to maximal electroshock. How- (b) ever, much delayed onset and duration of convulsion was witnessed at Fig. 5. Effects of N. laevis extracts on: (a) carrageenan and (b) formalin-induced medium dose (200 mg/kg) root extract. The values obtained were sig- rat paw oedema. Results are mean ± SD (n = 5). Statistical significance dif- nificant (p ≤ 0.05) when compared to control group (Fig. 6). ference between control and treated was determined at p ≤ 0.05 (one-way ANOVA followed Duncan’s multiple range test). 4. Discussion Table 7 In recent times, pharmacognosy has rapidly improved the use of Anticonvulsant effects of N. laevis extracts on strychnine-induced seizure in rats. medicinal plants for treating various diseases because of much Group dose Time to seizure onset Latency of deaths % advancement in technology such as chromatographic processes (like (s) (s) protection thin layer, paper, column, gas, liquid and high-performance chroma- 10 mL distilled 60.23 2.11 82.14 4.22 0 tography), microscopy, isolation and purification techniques. These ± ± water processes have made it possible the rapid isolation of bioactive com- 2 mg/kg diazepam no seizures no seizures 100 pounds that were thought to be difficult to isolate in their pure forms 100 mg/kg LE 102.10 ± 2.02 128.02 ± 2.01 40 previously. Pharmacognosy as an aspect of ethnobotany, pharmacology, 200 mg/kg LE 116.14 ± 4.012* 132.24 ± 4.02* 60 biological sciences, biochemistry, and medicinal chemistry has also 400 mg/kg LE no seizures no seizures 100 100 mg/kg RE 65.18 ± 1.01 76.12 ± 1.01 0 combined other spectroscopic techniques like NMR and FTIR to accu- 200 mg/kg RE 80.34 ± 2.04* 102.22 ± 4.02* 60 rately elucidate the structures of isolated compounds from medicinal 400 mg/kg RE no seizures no seizures 100 plants (Ukwubile et al., 2019b). These is very crucial in quality assur- Results are mean ± SD (n = 5),*statistical significance at p ≤ 0.05 (one-way ance of ethnomedicinal prescriptions from plants. It is important to note ANOVA followed Dunnette’s post-hoc), LE: leaf extract, RE: root extract. 35 3.4. Evaluation of anti-convulsant effects of N. laevis extracts 30 25 20 3.4.1. Strychnine-induced convulsion in rats 15 The results showed that at lower dose of extract administration to the 10 Onset of convulsion rats 40% protection was offered to the animals by leaf extract while the 5 0 Duration of convulsionroot extract does not offer any protection. More protection from seizure was offered at 400 mg/kg dose of leaf and root extracts. These results were significantly different (p ≤ 0.05) when compared to the control groups (Table 7). Group Fig. 6. Effects of N. laevis extracts on maximum electroshock-induced convul- sion in rats. Results are mean ± SD (n = 5), LE: leaf extract, RE: root extract. 7 Paw diameter ( SD) Paw oedema diameter ( SD) Time (min) C.A. Ukwubile et al. J o u r n a l o f E t h n o p h a r m a c o l o g y 314 (2023) 116632 these processes developed have also helped to establish authentic fin- and Sadiq, 2016). gerprints of medicinal plants’ products which is aimed preventing and For proper structural elucidation of isolated compounds from plant detecting adulteration of crude drugs. Furthermore, most of these iso- extract, the GC-MS has been recommended since it give mass-charge (m/ lated bioactive compounds have been used as lead compounds for the z) number of the compounds (Mohamad et al., 2018). From the current development of various known conventional medicines such as anti- study, methanol leaf extract of N. laevis contain important compounds cancer agents, antimalarials, antibiotics, antihypertensive agents, such as pyridine-3-yl-ethaniminidamide (m/z:135.1 g/mol), melibiose nutraceuticals, and antidiabetics (Belwal et al., 2014). Thus, medicinal (m/z: 342.30 g/mol), 2-N-butylacrolein (m/z:112.17 g/mol) and 2-eth- plants have continued to be major source of raw materials for most ylyformanilide (m/z: 149.23 g/mol) among other compounds (Table 4). essential synthetic drugs globally, and more than 100 medicinal plants The study also showed that methanol root extract contains 2-furanme- are used worldwide for therapeutic purposes (Rosandy et al., 2013). thanamine (m/z: 97.12 g/mol), hydrazne-1,2-dimethyl (m/z: 60.09 In the current study, pharmacognostic studies were carried out on g/mol), fomepizole (m/z: 82.11 g/mol) and 2,6-nonadienal (m/z: the leaf and root of Newbouldia laevis plant, and other biological activ- 138.21), among others (Table 5). These compounds play crucial roles in ities such as analgesic, anti-inflammation and anticonvulsant activities the body. For instance melibiose is a precursor for raffinose an important of the extracts from the leaf and root of the plant were determined in carbohydrate, pyridine-3-yl-ethaniminidamide and pyridine containing Wistar rats. The quantitative microscopical evaluation of the leaf drugs are used as anticancer, antimicrobial, antioxidant, antiviral, showed that it contains paracytic type of stomata that are hypostomatic antidiabetic, antihypertensive, anti-inflammatory and antimalarial (i.e., deeply situated) as viewed using the scanning electron microscope agents, as well as antiprotozoal agent and psychopharmacological an- (Fig. 1, Table 1). These stomata were numerous at the upper surface of tagonists (Keskes et al., 2017). Also, hydralizine-1,2-dimethyl isolated the leaf than the lower surface. This uneven distribution of stomata in from root extract has been reported also, as antihypertensive agent leaves of plants is of taxonomic and adaptation significance in plants. (lowering blood pressure), its roles in the current study was unknown. This is because, high number of stomata found on the upper surface help Similarly, fomepizole isolated from the root extract has been reported to to facilitate rapid transpiration of water from leaves thereby, preventing be used antidote for removing poisons from ethylene glycol or methanol, the cell from undergoing cellularly imbalance. This adaptive strategy and sometimes combined with hemo-dialysis to remove poisons from observed in this plant in the current study indicate that the plant is able the body (Ukwubile et al., 2019). to survive drought and desiccation withing the environment (Jones Pharmacognosy as an aspect of natural product science has made it et al., 2006). Trichomes carryout several functions in plant such as possible to assess the safety of ethnomedicinally used herbal prepara- maintaining still air on leaf surface and secretory of certain substances tions. In the current study, toxicity study showed that the extracts are that are of importance in pharmaceutical industries. In the present safe at the dose of 2000 mg/kg administered to the rats (Table 6). There study, the glandular unicellular trichomes observed in the leaf may serve were no signs of toxicity witnessed in the animals after fourteen days of same function especially secretory of sugary substances as seen in the acute oral toxicity testing, thus, justifies its use in traditional medicine plant parts like the stem, flowers, and fruits. for treating various diseases such as analgesic, anti-inflammation and Our study also revealed that the transvers section of the leaf and root anticonvulsant agents. For instance, our study from the acetic acid- (Fig. 2 a and b) showed that the leaf contain palisade and spongy pa- induced writhing analgesic test in rats revealed that the extracts renchyma, thin cuticle, calcium oxalate, and fiber sclereids with narrow significantly inhibited abdominal contraction induced by acetic acid in lumen in root. The presence of palisade and spongy parenchyma is of dose-dependent fashion with much inhibition witnessed in groups taxonomic significance in that it helps to distinguish the plant from treated with leaf extract (Fig. 3 and b). This implies that the extracts closely related species from other plant families or even the same family might have displayed their analgesic activities using the peripheral (Bignoniaceae). Moreover, palisade parenchyma has been reported as nervous system as compared to the standard drug (pentazocine) a clas- storage for sugar and starch while the spongy parenchyma helps to sical example of non-steroidal anti-inflammatory drugs (NSAIDs) used in facilitate gaseous exchange in plants (Dodiya and Jain, 2017). These this study. Similarly, in the tail-flick analgesic test, there was a dose- roles played by parenchyma is similar in this current study because of dependent reaction time response. The study showed that the combi- the ability of the plant to withstand long drought and desiccation. nation of naloxone and 400 mg/kg leaf extract displayed significant Physicochemical evaluations are very crucial in pharmacognostic delayed in reaction time when compared to naloxone and pentazocine profiling of medicinal plants. This is because it helps to establish accu- group which indicates the involvement of opioid receptors (Fig. 4a and rate fingerprints for crude drugs that could be use as important tool for b). It implies that the extracts exhibited analgesic action mediated creating monographs on drugs (Kunle, 2012). In this study, moisture peripherally and centrally by involving the opioid receptors because contents were 6.25 ± 0.01% w/w and 8.04 ± 0.10% w/w for leaf and naloxone was considered as a non-selective opioid receptor antagonist root respectively (Table 2). It showed that root powders are likely to be with a short duration of pharmacological actions (Hijazi et al., 2017). exposed to microbial attack than the leaf powder, thus, it should be From the results obtained in anti-inflammatory study by properly stored in dry and clean environment to prevent microbial carrageenan-induced paw oedema in rats, the extracts showed dose- contamination. Similarly, water extractive value of leaf (10.28 ± 1.02% dependent inhibition of paw oedema with increasing experimental w/w) was greater than that of the root (6.20 ± 0.01% w/w). This results times. However, many reductions in paw volumes were obtained with was similar to previous report obtained other researchers where it was rats treated with N. laevis leaf extract (Fig. 5 a and b). The use of reported that water extractive value was greater than alcohol extractive carrageenan to induce inflammation in the rats triggered the release of values (Bijauliya et al., 2021). The present phytochemical study further mediator of inflammation such as serotonin, bradykinin and histamine revealed that tannins, flavonoids, alkaloids, triterpenes and saponins which are released at the onset of inflammation followed by prosta- were the major secondary metabolites detected in leaf extract while the glandins which are released in the later stage of inflammation (Pandey root extract also contain cardiac glycosides in addition to other phyto- et al., 2020). Similarly, a dose-dependent reductions in paw oedema constituents (Table 3). It is an established fact that these phytochemicals were obtained in formalin-induced inflammation in rats (Fig. 5 b). The play crucial roles in various aspects of human healthcare system such as ability of the plant extracts to greatly inhibit the inflammatory action of anticancer, antimalarials, antidiabetics, antibiotics, antihypertensive, other promotors of inflammation such as tumor necrosis factor-A, and anti-inflammatory agents (Omoregie et al., 2010). Their roles in the cyclooxygenase-2 (COX-2) and interleukins which further affirms their present study were not different from the previously reported ones. use as anti-inflammatory prescription in traditional medicine. Many of the family Bignoniaceae have been reported to contain various In the current study, strychnine-induced convulsion in rats showed types of flavonoids and triterpene that are used as treatment for dose-dependent protection of the animals and delayed onset of seizures inflammation and malaria respectively in traditional medicine (Bairagi (Table 7). Strychnine is an alkaloid which is isolated from dried ripped 8 C.A. Ukwubile et al. J o u r n a l o f E t h n o p h a r m a c o l o g y 314 (2023) 116632 seed extract of Strychnos nux vomica, a shrub found mainly in East Indies Data availability which has been used as CNS stimulant among other uses. In addition, the use of Pentylenetetrazole (PTZ), a GABA-A receptor antagonist Data will be made available on request. conferred 100% protection on the animals especially 100 and 400 mg/ kg leaf and root extract doses (Table 8). The plant extracts reverse the Acknowledgements symptoms of convulsions and seizures even death which was caused by PTZ by modulation of glutamatergic neurotransmitter. The result was The authors are thankful to Mr. Suleiman Mejida of the Central not different from what was obtained in the maximal electroshock (MES) Research Laboratory, University of Lagos, Nigeria for assisting in the GC- model where there was significant delay in the onset of convulsion in MS analysis, Mr. Yusuf of the Pharmacology and Toxicology Laboratory, animals given leaf extract than root extract (Fig. 6). The MES induced University of Maiduguri, Nigeria for helping in some laboratory pro- convulsion model activate the release of calcium and sodium pump cedures and Dr. Imran Janmohammed of Anthias Consultant, United channels, making the extracts to be able to prevent the flow of these ions Kingdom, for his technical assistance. which subsequently resulted in delayed onset of convulsion and seizures witnessed. In general, most anticonvulsant drugs that are used clinically References today exert their actions by blockage of neuronal ion channels or facil- itating the neurotransmission of gamma-aminobutryic acid (GABA) Akhigbemen, A.M., Ozolua, R.I., Bafor, E.E., Okwuofu, E.O., 2019. Evaluation of some (Shinde et al., 2018). Our study showed that the anticonvulsant activity neuropharmacological effects of Caladium bicolor aiton (araceae) leaf extracts in mice. Metab. Brain Dis. 34 (2), 537–544. https://doi.org/10.1007/s11011-019- of leaf and root extract of N. laevis might have been exhibited following 0390-z. any of the mention mechanisms or both. Alam, F., Din, K.M., Rasheed, R., Sadiq, A., Jan, M.S., Minhas, A.M., Khan, A., 2020. Phytochemical investigation, anti-inflammatory, antipyretic and antinociceptive activities of Zanthoxylum armatum DC extracts-in vivo and in vitro experiments. 5. Conclusion Heliyon 6 (11), e05571. https://doi.org/10.1016/j.heliyon.2020.e05571. Alamgir, A.N.M., 2017. In: Alamgir, A.N.M. (Ed.), Introduction BT - Therapeutic Use of Medicinal Plants and Their Extracts: Volume 1: Pharmacognosy, 17. Springer Our study revealed important pharmacognostic profiles that will International Publishing, p. 1. https://doi.org/10.1007/978-3-319-63862-1_1. help in both taxonomic identification of the plants’ leaf and root, and Anes, U.C., 2015. Antinociceptive and anti-inflammatory properties of Vernonia pharmaceutical indices for building drug monographs. The study also pauciflora Willd. (Asteraceae) ethanol extracts. J. Pharmacogn. Phytochem. 7 (6) showed that methanol leaf and root extracts of N. laevis showed dose- https://doi.org/10.5897/JPP2012.061. Anes, C., Salvia, T., Oise, E., Modu, B., Amarachukwu, V., 2023. Evaluation of dependent analgesic, anti-inflammatory, and anticonvulsant activities. physicochemical parameters , acute and subchronic toxicities , and anti-diabetic These activities are due the presence of certain phytochemicals and activity of Spondias venulosa (Engl .) Mart . ex Engl . leaf extract on alloxan-induced bioactive compounds in leaf and root extracts. The study further diabetic rats. J. Ethnopharmacol. 306 (December 2022), 116169 https://doi.org/ 10.1016/j.jep.2023.116169. revealed that anticonvulsant activity of the plant was achieved by the Bairagi, S., Sadiq, M.A.H., 2016. Invasive toad ’ S secretions acts. As Anticancer Agents [a blockage of neuronal ion channels or promotion of GABAergic neuro- Review] 5 (3), 518–526. transmission release. Our study further affirmed the use of N. laevis as Belwal, C., Goyal, P.K., Balte, A., Kolhe, S., Chauhan, K., Rawat, A.S., Vardhan, A., 2014. Isolation, identification, and characterization of an unknown impurity in lovastatin. ethnopharmacological prescription for pains, inflammation and EP. Sci. Pharma 82 (1), 43–52. https://doi.org/10.3797/scipharm.1305-04. convulsion or epilepsy in traditional medicine in Nigeria. Bijauliya, R.K., Kannojia, P., Mishra, P., Singh, P.K., Kannaujia, R., 2021. Pharmacognostical and physiochemical study on the leaves of nyctanthes arbor- tristis linn. J. Drug Deliv. Therapeut. 11 (4), 30–34. https://doi.org/10.22270/jddt. Ethical approval v11i4.4914. Dermane, A., Kpegba, K., Eloh, K., Osei-Safo, D., Amewu, R.K., Caboni, P., 2020. The rats used in the present study was approved for the use with Differential constituents in roots, stems and leaves of Newbouldia laevis Thunb. screened by LC/ESI-Q-TOF-MS. Res. in Chem. 2, 100052 https://doi.org/10.1016/j. approval obtained from the Animal in Research Ethical Group of PJ Rats rechem.2020.100052. Farm Ltd with approval number: PJF/NG/JOS-035-2023. Dighe, A.P., Barve, K.H., 2019. Anticonvulsant effect of Sphaeranthus flower extracts in mice. J. Ayurveda Integr. Med. 10 (1), 38–40. https://doi.org/10.1016/j. jaim.2018.06.008. CRediT authorship contribution statement Dodiya, T., Jain, V., 2017. Pharmacognostic, physicochemical and phytochemical investigation of Grangea maderaspatana. J. Phytopharmacol. 6 (6), 359–363. Cletus Anes Ukwubile: Designed the project, performed the https://doi.org/10.31254/phyto.2017.6611. Eiceman, G.A., Hill, H.H., Davani, B., 1994. Gas chromatography. Anal. Chem. 66 (12), experiment, Formal analysis, and preparation of the manuscript. 621–633. https://doi.org/10.1021/ac00084a023. Emmanuel Oise Ikpefan: Performed the experiment, review the liter- Hijazi, M.A., El-Mallah, A., Aboul-Ela, M., Ellakany, A., 2017. Evaluation of analgesic ature, and, supervised the project. Musa Yusuf Dibal: Performed the activity of papaver libanoticum extract in mice: involvement of opioids receptors. Evi. Compl. and Alter. Med. 2017 https://doi.org/10.1155/2017/8935085. experiment, data analysis, and, literature review. Vivian Amar- Iwu, M.M., 2014. Handbook of African Medicinal Plants, second ed. CRC, Press Ltd, Boca achukwu Umeano: Formal analysis, Data curation, Performed the Raton New York. Taylor & Francis Inc. experiment, data analysis and literature review. David Nnamdi Men- Jones, P.W., Chin, Y.-W., Kinghorn, D.A., 2006. The role of pharmacognosy in modern medicine and pharmacy. Curr. Drug Targets 7 (3), 247–264. https://doi.org/ kiti: Formal analysis, Writing – review & editing, Performed the writing 10.2174/138945006776054915. – review and editing and performed the experiment. Clement Chidi Keskes, H., Belhadj, S., Jlail, L., El Feki, A., Damak, M., Sayadi, S., Allouche, N., 2017. Kaosi: Performed the experiment and review the literature. Simon LC-MS-MS and GC-MS analyses of biologically active extracts and fractions from tunisian juniperus phoenice leaves. Pharm. Biol. 55 (1), 88–95. https://doi.org/ Paul: Formal analysis, Data analysis, and, Supervision, supervision of 10.1080/13880209.2016.1230139. the project. Ademola Clement Famurewa: Performed the experiment, Khalifa, M.A., 2022. Original Article Acute and Subacute Toxicity Studies of, vol. 8, data analysis, and, Supervision, supervision of the project. Henry Net- pp. 279–290, 4. Kim, G.S., Zeng, L., Alali, F., Rogers, L.L., Wu, F.E., McLaughlin, J.L., Sastrodihardjo, S., tey: Formal analysis, Supervision, supervision of the project, Writing – 1998. Two new mono-tetrahydrofuran ring acetogenins, annomuricin E and review & editing, Review and editing of the manuscript. Timothy muricapentocin, from the leaves of Annona muricata. J. Nat. Prod. 61 (4), 432–436. Samuel Yerima: Formal analysis, Review of manuscript, and, Supervi- https://doi.org/10.1021/np970534m. sion, supervision of the project, All authors approved the final manu- Kumar, T., Gupta, A., Gidwani, B., Kaur, C.D., 2015. Phytochemical screening and evaluation of anthelmintic activity of Euphorbia tithymaloidus. Int. J. Biol. Chem. 9 script before submission. (6), 295–301. https://doi.org/10.3923/ijbc.2015.295.301. Kunle, 2012. Standardization of herbal medicines - a review. Int. J. Biodivers. Conserv. 4 (3), 101–112. https://doi.org/10.5897/ijbc11.163. Declaration of competing interest Lalthanpuii, P.B., Lalchhandama, K., 2020. Phytochemical analysis and in vitro anthelmintic activity of Imperata cylindrica underground parts. BMC Compl. Med. and Ther. 20 (1), 1–9. https://doi.org/10.1186/s12906-020-03125-w. We have none to declare. 9 C.A. Ukwubile et al. J o u r n a l o f E t h n o p h a r m a c o l o g y 314 (2023) 116632 Lee, Y.M., Son, E., Kim, S.H., Kim, O.S., Kim, D.S., 2019. Anti-inflammatory and anti- Prior, R.L., Wu, X., Schaich, K., 2005. Standardized methods for the determination of osteoarthritis effect of Mollugo pentaphylla extract. Pharm. Biol. 57 (1), 74–81. antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food https://doi.org/10.1080/13880209.2018.1557700. Chem. 53 (10), 4290–4302. https://doi.org/10.1021/jf0502698. Mante, P.K., Adongo, D.W., Woode, E., Kukuia, K.K.E., Ameyaw, E.O., 2013. Rasul, Mohammed Golam, 2018. Extraction, isolation and characterization of natural Anticonvulsant Effect of Antiaris Toxicaria (Pers.) Lesch. (Moraceae) Aqueous products from medicinal plants. Intl. J. Basic Sci. Appl. Comput. 2 (6), 1–6. Extract in Rodents. ISRN Pharmacology, 519208. https://doi.org/10.1155/2013/ Rosandy, A.R., Din, L.B., Yaacob, W.A., Yusoff, N.I., Sahidin, I., Latip, J., Nataqain, S., 519208, 2013. Noor, N.M., 2013. Isolation and characterization of compounds from the stem bark Mohamad, S., Ismail, N.N., Parumasivam, T., Ibrahim, P., Osman, H., Wahab, H.A., 2018. of Uvaria rufa (Annonaceae) Pemisahan dan Pencirian sebatian Dari Kulit batang Antituberculosis activity, phytochemical identification of Costus speciosus (J. Uvaria rufa (Annonaceae). Mala. J. Analy. Sci. 17 (1), 50–58. Koenig) Sm., Cymbopogon citratus (DC. Ex Nees) Stapf., and Tabernaemontana Sharma, V.C., Kaushik, A., Dey, Y.N., Srivastava, B., Wanjari, M., Jaiswal, B., 2020. coronaria (L.) Willd. and their effects on the growth kinetics and cellular integrity of Analgesic, anti-inflammatory and antipyretic activities of ethanolic extract of stem Mycobacteri. BMC Compl. Alternative Med. 18 (1), 1–14. https://doi.org/10.1186/ bark of Anogeissus latifolia Roxb. Clin. Phytosci. 6 (1) https://doi.org/10.1186/ s12906-017-2077-5. s40816-020-00171-2. Nainwani, R., Gupta, A., Batra, M., & Hardik, P., n.d. Anti-Inflammatory Activity of Shelar, M.K., Patil, M.J., Bhujbal, S.S., Chaudhari, R.B., 2018. Evaluation of Various Fractions of Methanolic Extract of Punica Granatum Rind with its anticonvulsant activity of the ethanolic extracts from leaves of Excoecaria agallocha. Phytochemical Evaluation Pubicon.. Future J. Pharm. Sci. 4 (2), 215–219. https://doi.org/10.1016/j.fjps.2018.06.002. Ndidi, C., Ngozika, F., Amaka, P., Blessing, N., 2020. Phytochemical and antibacterial Shinde, M., Gilhotra, R., Chaudhari, S., 2018. Anticonvulsant and sedative activities of activities of Vernonia amygdalina leaves (bitter leaf) on two drug resistant bacteria. extracts of carissa carandas leaves. J. Drug Deliv. Therapeut. 8 (5), 369–373. https:// Intl. J. Res. Stud. Microbiol. Biotech. 6 (1) https://doi.org/10.20431/2454- doi.org/10.22270/jddt.v8i5.1934. 9428.0601004. Ukwubile, C.A., 2017. Preliminary phytochemical screening and antibacterial activity of Nonglang, F.P., Khale, A., Bhan, S., 2022. Phytochemical characterization of the thaumatococcus daniellii (benn.) benth. (Marantaceae) leaf extract. J. Bacteriol. ethanolic extract of Kaempferia galanga rhizome for anti-oxidant activities by Mycol.: Open Access 4 (2), 2–6. https://doi.org/10.15406/jbmoa.2017.04.00086. HPTLC and GCMS. Future J. Pharm. Sci. 8 (1), 1–12. https://doi.org/10.1186/ Ukwubile, C.A., Ahmed, A., Katsayal, U.A., Ya’u, J., Mejida, S., 2019a. GC–MS analysis of s43094-021-00394-1. bioactive compounds from Melastomastrum capitatum (Vahl)Fern. leaf methanol Obese, E., Biney, R.P., Henneh, I.T., Adakudugu, E.A., Anokwah, D., Agyemang, L.S., extract: an anticancer plant. Sci. Afri 3, 10–17. https://doi.org/10.1016/j. Woode, E., Ameyaw, E.O., 2021. The anticonvulsant effect of hydroethanolic leaf sciaf.2019.e00059. extract of calotropis procera (ait) R. Br. (Apocynaceae). Neural Plast., 5566890 Ukwubile, C.A., Ahmed, A., Katsayal, U.A., Yau, J., Nettey, H.I., 2019b. Acute and https://doi.org/10.1155/2021/5566890, 2021. subchronic toxicity assessment of Melastomastrum capitatum Fern. leaf methanol Olaleye, O.O., Oladipupo, A.R., Oyawaluja, B.O., Herbert, A.B., 2021. Chemical extract in Wistar albino rats. Intl. Biol. Biomed. J. 5 (3), 0, 0. http://ibbj.org/arti Composition , Antioxidative and Antimicrobial Activities of Different Extracts of the cle-1-232-en.html. Leaves of Parquetinanigrescens (Asclepiadaceae), vol. 4, pp. 359–371, 4. Yadav, Abhishek, Samanta, K., 2021. Formulation and evaluation of herbal ointment Olatokunboh, A.O., Kayode, Y.O., Adeola, O.K., 2009. Anticonvulsant activity of using Emblica officinalis extract. World J. Adv. Res. Rev. 9 (2), 32–37. https://doi. rauvolfia vomitoria (afzel). Afri J. Pharm. Pharmacol. 3 (6), 319–322. org/10.30574/wjarr.2021.9.2.0040. Omoregie, H., Emmmanuel, O.O., Grace, U., Sabo, M., Samuel, E., Folashade, O., Yeddes, W., Mejri, I., Grati-Affes, T., Khammassi, S., Hammami, M., Aidi-Wannes, W., Ibrahim, J., Ibekwe, N., Ibumeh, J., 2010. Phytochemical screening and Saidani-Tounsi, M., 2022. Synergistic effect of cloves (syzygium aromaticum), thyme antimicrobial studies of methanol , ethyl acetate and hexane extracts of vitex (thymus vulgaris) and lemon (citrus limon) blended essential oils optimized by doniana. Sweet . (Stem Bark and Leaf) 8 (8), 177–185. mixture design for improving the antioxidant activity. Avi. J. Med. Biochem. 10 (1), Pages, A., Agronomique, R., Brab, L., 2013. Institut National des Recherches Agricoles du 20–29. https://doi.org/10.34172/ajmb.2022.03. Bénin (INRAB) Sommaire, vol. 229, pp. 27–35. Zhang, F., Xu, Y., Pan, Y., Sun, S., Chen, A., Li, P., Bao, C., Wang, J., Tang, H., Han, Y., Pandey, B.P., Adhikari, K., Pradhan, S.P., Shin, H.J., Lee, E.K., 2020. Inflammatory 2019. Effects of angiotensin-(1-7) and angiotensin II on acetylcholine-induced activities of selected medicinal plants from western Nepal. Future J. Pharm. Sci. 6, vascular relaxation in spontaneously hypertensive rats. Oxid. Med. Cell. Longev. 1–12. https://doi.org/10.1155/2019/6512485, 2019. 10