Pharmacognostic Study and Hepatoprotective Activity of the Methanolic Extract and Fractions of Leaves of Picralima nitida Apocyanaceae

1.        Introduction

Liver is the largest organ, accounting for approximately 2% to 3% of average body weight (1). It functions as a centre for metabolism of nutrients, excretion of waste metabolites and controls the flow and safety of substances absorbed from the digestive system before distribution to the systemic circulatory system (2,3). According to the World Health Organization, an estimated 354 million people were reported to be living with hepatitis infection and for most, testing and treatment remain beyond reach (4). The symptoms of liver disease may include jaundice, abdominal pain and swelling, swelling in the legs and ankles, itchy skin, dark urine colour among other signs.

 Researches on plants have shown that plants harbour in them bioactive phytochemicals (5–7) and from plants have been isolated phytocompounds (7). Picralima nitida (Stapf) also known as the Akuamma plant is found in tropical African countries such as Ivory Coast, Nigeria, Uganda, and Gabon (8). It is popularly known as Abeere in the Southwestern part of Nigeria among the Yoruba people (9–11). The plant is known for its medicinal purposes and is used in traditional medicine for the treatment and management of diseases such as malaria, abscesses, hepatitis, pneumonia, diabetes, and hypertension (10–12). The previous works done on the leaves, stem bark, fruits, seeds and pods of P. nitida revealed polyphenols, peptide, amide, ester, terpenoids, and indole, alkaloids; akuammine, akuammicine, akuammidine and akuammiline as major compounds (10). In a study by De Campos et al. 2020 aqueous seed extract of P. nitida was shown to alleviate dyslipidaemia, hyperglycaemia, and pro-oxidant status associated with the intake of a high-fat high fructose diet (13). P. nitida leaf extract has been shown to ameliorate oxidative stress and modulates insulin signalling pathway in high fat-diet/STZ-induced diabetic rats (14). A study has also shown that Picralima nitida seed and pod have hepatoprotective activity at 400 mg/kg once daily for 14 days in CCl4 induced liver damage or injury in animal model (15).

Figure 1 Picralima nitida

This study aims to undertake the pharmacognostic analysis and evaluate the methanol extract and fractions of the leaf on the potential hepatoprotective effect against carbon tetrachloride (CCl₄) induced liver damage in Swiss albino mice.

2.        Materials and Methods

2.1     Chemicals

Chemicals and experimental reagents used include methanol, n-Hexane, butanol, ethyl acetate, diethyl ether (JHD, China), Tween-80, Ascorbic acid, Fehling’s solution (A&B), Ammonia solution, Millions reagent, ferric chloride (Griffin & George, England), thiobarbituric acid (TBA) (Guangdong Guanghua Chemical Factory Co., Ltd, China), HCL, Alkaline phosphatase reagent kit (Teco Diagnostics, USA), Aspartate aminotransferase reagent kit (Randox Laboratories limited, United Kingdom), Alanine aminotransferase reagent kit (ALT, Randox Laboratories Limited, United Kingdom), etc. All solvents/reagents purchased were of analytical grade. All laboratory reagents were freshly prepared and freshly distilled water was used when required.

2.2     Animals

Swiss albino mice (25–30 g) were employed for the study. All the animals were obtained from the Animal House of the Department of Pharmacology and Toxicology, Enugu State University of Science and Technology, Enugu State. The animals were housed in standard laboratory conditions. The animals were allowed free access to food and water and all animal experiments were conducted in compliance with the NIH guide for the care and use of laboratory animals (National Institute of Health (NIH) (2011) Pub No: 85-23). Institutional animal ethics approval was obtained (ESUT/AEC/0138/AP096).

2.3     Collection of Plant Material

The leaves of P. nitida were purchased in July 2021 from Ibadan in Oyo State, Nigeria. The plant was identified and authenticated by a taxonomist Mr Felix Nwafor at the department of Pharmacognosy and Environmental Science, University of Nigeria, Nsukka, and the herbarium specimen was deposited at the University of Nigeria, Nsukka, Enugu State, Nigeria, with voucher number PCG/UNN/0442.

2.4     Preparation and Extraction of Plant Material

The leaves of P. nitida collected were cleaned to remove contaminants and air dried under room temperature. They were further pulverized to a fine powder using a mechanical grinding machine. The powdered leaves were stored in an air-tight container till further use. A 1.5 kg amount of the powder was extracted in 4.5 L of 99% methanol by cold maceration for 72 hours with intermittent shaking. The solutions were filtered with Whatman filter paper and the filtrates obtained were concentrated using rotary evaporator at 40°C. 

2.5     Fractionation of Plant Extract

The crude methanol extract of P. nitida (107.68 g) was subjected to liquid-liquid partition successively with n-hexane, butanol, ethyl acetate and water in increasing order of polarity to obtain n-hexane, butanol and ethyl acetate and water fractions respectively. The fractions were concentrated using rotary evaporator at 40°C.

2.6     Microscopy Evaluation

The qualitative and quantitative microscopy was done according to the method described by Nwafor et al. (2019).  The Freehand section of the leaves was prepared by clearing method and stained with safranin solution to reveal the epidermal cells, stomata type and size, stomata density and index, trichome parameters and vein islet numbers. They were viewed under a light phase contrast microscope (Motic B3, Motic Carlsbad, CA, USA) at x 40, x 100, and x 400 magnifications and photomicrographs were taken with a Moticam 2.0 image system with software (Motic Carlsbad, CA, USA). All parameters were observed on both the adaxial and abaxial surfaces of the leaves. A chemomicroscopy examination was also conducted on the leaf powder to determine the presence of starch, calcium oxalate crystals, and lignified vessels using standard methods).

2.7     Physicochemical Studies

The physicochemical analysis of the leaf powder was carried out to determine the total ash, acid-insoluble ash, water-soluble ash and extractive value using standard methods (16).

2.8     Acute Toxicity

Acute toxicity tests were performed in mice according to the method described by Lorke (17).

2.9     Experimental Design

Sixty (60) Swiss albino mice were divided into seven groups. Groups one to five have ten (10) mice each, while groups six and seven have 5 mice each. Five (5) of the mice in group one were pre-treated with 200 mg/kg of the methanol extract, while five (5) were pre-treated with 400 mg/kg of the methanol extract. Five (5) of the mice in group two were pre-treated with 200 mg/kg of the n-hexane fraction, while five (5) were pre-treated with 400 mg/kg of the n-hexane fraction. Five (5) of the mice in group three were pre-treated with 200 mg/kg of the ethyl acetate fraction, while five (5) were pre-treated with 400 mg/kg of the ethyl acetate fraction. Five (5) of the mice in group four were pre-treated with 200 mg/kg of the butanol extract, while five (5) were pre-treated with 400 mg/kg of the butanol fraction. Five (5) mice in group five were pre-treated with 200 mg/kg of the water fraction, while five (5) were pre-treated with 400 mg/kg. Group six (6) was pre-treated with 100 mg/kg of ascorbic acid (positive control), while group seven (7) served as the negative control (2 % tween 80 and 80 ml/kg of water). After 14 days, the animals in all the groups except those in the negative control group were administered CCl₄ (49 ml dissolved in 1 ml of olive oil) through intraperitoneal injection. Blood samples were collected from all the animals through the orbital sinus after 24 hours. They were centrifuged (model 7GL-20M, China) at 3000 rpm for 10 minutes, and the supernatant was decanted to get the serum. The serum was used to estimate the serum liver marker enzymes, which are Alkaline phosphatase (ALP), Aspartate aminotransferase (AST), Alanine transaminase (ALT), and lipid peroxidation (MDA)

2.10  Statistical Analysis

The results were analysed using SPSS version 16 and presented as mean ± standard error of mean (SEM). Significance between control and extract-treated groups were determined using one-way analysis of variance (ANOVA). Differences between means were considered statistically significant at P < 0.05.

3.        Results

3.1    Yield of P. nitida Methanol Extract and Fractions

  The yield in gram and percentage of the methanolic leaves extract and fractions of P. nitida are presented in Table 1.

Table 1 The Yields of Methanol Extract and Fractions of P. nitida leaf

^aYield calculated from 1250 g of powdered leaves, ^bYield calculated from 87.58 g of methanol extract

3.2   Fresh leaf microscopic analysis of P. nitida

 The result of the fresh leaf microscopic examination of the fresh leaf of P. nitida is present in Figures 1 and 2. The transverse section of the leaf is presented in Figure 3, showing the upper epidermis, palisade tissue, collenchyma, xylem, lower epidermis and phloem. Presented in Figure 4 is the Chemomicroscopy of the powder showing reticulate type of vessel elements aligned with fibre and parenchyma cells. The vessel and fibre elements are lignified while the ray parenchyma is not lignified. Presented in Figure 5 is the Photomicrograph of the powdered leaf of P. nitida showing a pack of palisade tissue, prism-shaped calcium oxalate crystal and isolated and coiled fibre elements. Presented in Table 2 and 3 are the results of the quantitative and qualitative microscopy respectively.

Figure 1 Adaxial (upper) surface of the leaf of P. nitida showing polygonally-shaped epidermal cells

Figure 2 Abaxial (lower) surface of the leaf of P. nitida showing polygonally-shaped epidermal cells. Stomata are present (paracytic type). Trichomes are absent

Figure 3 Transverse section of midrib of leaf of P. nitida

Figure 4 Chemomicroscopy of powdered leaf of P. nitida 

Figure 5 Photomicrograph of the powdered leaf of P. nitida 

   Table 2 Quantitative microscopic result