Antioxidant and cytotoxicity assessment of the methanolic extract of Annona reticulata Linn

Medicinal plants are globally valuable sources of herbal products, and they are disappearing at a high speed. This article reviews global trends, developments and prospects for the strategies and methodologies concerning the conservation and sustainable use of medicinal plant resources to provide a reliable reference for the conservation and sustainable use of medicinal plants. We emphasized that both conservation strategies (e.g. in situ and ex situ conservation and cultivation practices) and resource management (e.g. good agricultural practices and sustainable use solutions) should be adequately taken into account for the sustainable use of medicinal plant resources. We recommend that biotechnical approaches (e.g. tissue culture, micropropagation, synthetic seed technology, and molecular marker-based approaches) should be applied to improve yield and modify the potency of medicinal plants [1, 2]. Medicinal plants are globally valuable sources of new drugs. There are over 1300 medicinal plants used in Europe, of which 90 % are harvested from wild resources; in the United States, about 118 of the top 150 prescription drugs are based on natural sources. Furthermore, up to 80 % of people in developing countries are totally dependent on herbal drugs for their primary healthcare, and over 25 % of prescribed medicines in developed countries are derived from wild plant species. With the increasing demand for herbal drugs, natural health products, and secondary metabolites of medicinal plants, the use of medicinal plants is growing rapidly throughout the world [3, 4].  The presence of active substances like alkaloids, flavonoids, glycosides, vitamins, tannins, and coumarin compounds in the organs of medicinal plants influences their therapeutic properties. These substances have a physiological impact on both human and animal bodies or are biologically active in relation to the diseases they are used to treat [5]. Plants can provide biologically active molecules and lead structures for the development of modified derivatives with enhanced activity and reduced toxicity. Medicinal plants are considered as a rich resources of ingredients which can be used in drug development either pharmacopoeial, non- pharmacopoeial or synthetic drugs. Along with medicinal formulations plants have been successfully utilised for the development of cosmetics and toiletry preparations [6].  Plant-derived products have an imperative biological role against certain pathogenic organisms and were considered to be a major source of modern drugs. Rural people residing in developing countries are relying on traditional herbal medical system due to their strong believe and minimum access to allopathic medicines. Hence, ethnomedicinal knowledge is useful for the maintenance of community’s based approaches under this medical system. Present study was carried out in an unexplored remote tribal area of Pakistan to investigate and document the existing ethnomedicinal knowledge on local flora. Plant-derived products have an imperative biological role against certain pathogenic organisms and were considered to be a major source of modern drugs. Rural people residing in developing countries are relying on traditional herbal medical system due to their strong believe and minimum access to allopathic medicines. Hence, ethnomedicinal knowledge is useful for the maintenance of community’s based approaches under this medical system. Present study was carried out in an unexplored remote tribal area of Pakistan to investigate and document the existing ethnomedicinal knowledge on local flora [7]. This is proven by the fact that of the total number of publications analyzed, more than 100,000, only 11% are in the Agricultural and Biological Sciences category, while more than 50% are grouped in the Pharmacology, Toxicology and Pharmaceutics category and Medicine. This study highlights the scarce research from the agronomic perspective regarding domestication, production or genetic or biotechnological research on breeding of medicinal plants [8].  Plants contain abundant amount of secondary metabolites, they are considered to be principal source of therapeutically active compounds. Although all plants contain chemical constituents of one kind or another, all of them are not pharmacologically active. The chemical constituents, which are capable of influencing the physiological systems of the animal body by exerting some pharmacological actions, are designated as active chemical constituents of simply constituents [9]. These medicinally active chemical substances are generally produced in the plants as by-products or side products during the synthesis and metabolism of the primary metabolites and are not used or utilized by the plants for their normal life activities. They are termed as secondary metabolites. These compounds may be distributed in all parts of the plant body or may present in higher amount in some specific part [9].

Table 1. Chemical constituents of some medicinal plants [9].

The Annona genus (Annonaceae) consists of about 119 species, most of which are shrubs and trees widely distributed in the tropical and subtropical regions, including the Southeast Asia countries such as Malaysia, Indonesia, Thailand, Cambodia, Laos, and Vietnam [11]. A. reticulate L. is widely distributed in tropical and subtropical regions. The plant is indigenous to the West Indies. In India it is widely cultivated and naturalized as a fruit consuming plant and deciduous tree. It is distributed in Bengal, Burma and Southern regions of India. It is native to tropical regions of America, particularly in West Indies and South America. The plant is widely cultivated in Bangladesh and Pakistan [12]. Several species of Annona bear highly prized fruits. The alligator apple (4. glabra) of tropical America and western Africa, also known as pond apple and corkwood, is a 12-metre (40-foot) evergreen tree with 18- cm- (7-inch-) long oval leaves and fragrant yellowish flowers. It bears smooth, gnarled, yellowish fruits, 5-10 cm long, which are edible but of poor flavour. Its roots are used to make bottle corks and fishing floats and as rootstock for grafting less hardy species of Annona. A. reticulata is a small semideciduous tree reaching 6.0-7,5 m tall. It contains numerous lateral branches. It is a small tree with glabrous branches. The stems are cylindrical having lenticels and very short coffee-coloured hairs.

Figure 1. Leaves And Fruits Of Annona Reticulata

Annona reticulata Linn is a versatile tree and its fruits are edible. It possesses several medicinal properties such as anthelmintic, analgesic, anti-inflammatory, antipyretic, wound healing and cytotoxic effects. Traditionally the plant has been employed for the treatment of epilepsy, dysentery, cardiac problem, parasite and worm infestations, constipation, haemorrhage, bacterial infection, dysuria, fever, ulcer and as insecticide. Bark is a powerful astringent and used as a tonic whereas leaves used for helminthiasis treatment. The bark of the plant A. reticulata L. is a powerful astringent and given as tonic. The plant has been used as an anti-inflammatory agent in wound healing, anti-anxiety, anti-stress, anti- mutagenic, and spasmolytic agent. Leaf and stem extract shows inotropic, positive chronotropic and spasmolytic activities [13].

METHODS AND MATERIALS:

Collection and Identification:

First, a member of the Annonaceae family, Annona reticulata L., was chosen for this inquiry with the aid of a thorough literature review. The leaves were collected from Narail district, Bangladesh and identified by the taxonomist of the National Herbarium of Bangladesh, Mirpur, Dhaka. For future reference, the voucher specimens of the plants have been kept at the herbarium.

Plant Material Preparation:

To make the plant part (leaves) acceptable for grinding, they were first dried in the sun separately and then at a lower temperature (no higher than 50°C) in a hot air oven (Size 1, Gallenkamp). After that plant parts were ground into coarse powders in the Department of Pharmacy. Jahangirnagar University using high capacity grinding mill which were then stored in air-tight container with necessary markings for identification and kept in cool, dark and dry place for the investigation.

Extraction Procedure:

The Soxhlet equipment was used to extract the 200 g of powdered plant material using methanol at a temperature of 65 °C (1000 ml of solvent). The plant material was dried and used once more for the subsequent extraction after each extraction. In cycles of colorless liquid siphoning in the Soxhlet apparatus, it was confirmed that extraction was complete when the plant materials became depleted of their contents. The plant components were dried when the methanol extract was finished, then steeped in distilled water (IL). For seven days, the plant materials were kept in water in a sealed container with string and sporadic shaking. Each individual extract was filtered through a clean cotton bed. The acquired filtrates were dried at 40+2°C to produce a sticky concentration of the crude filtrate. The crude extract were used for phytochemical and pharmacological (in vitro) evaluation.

Phytochemical screening:

The crude plant extract were subjected to different qualitative tests to find out the presence of chemical constituents. These were identified by characteristic color changes using standard procedure.

Using the Folin-Ciocalteu Reagent (FCR), the total phenolic component concentration in plant methanolic extracts was ascertained as previously described. For the colorimetric testing of phenolic and polyphenolic antioxidants, phosphomolybdate and phosphotungstate are combined to create the Folin-Ciocalteu reagent (FCR), also known as Folin's phenol reagent or Folin-Denis reagent. It functions by calculating the concentration of the material under test that is required to prevent the reagent from oxidizing.

Flavonoids determination:

The colorimetric approach based on aluminum chloride has as its basic premise the formation of acid stable complexes between aluminum chloride and the C-4 keto group and either the C-3 or C-5 hydroxyl group of flavones and flavonols. Moreover, aluminum chloride and the ortho-dihydroxyl groups in the A- or B-ring of flavonoids combine to produce acid-labile complexes. The wavelength used to measure absorbance is 415 nm.

Total antioxidant capacity determination: Typically, the phosphor-molybdenum technique finds antioxidants like carotenoids, ?-tocopherol, ascorbic acid, and some phenolics. The antioxidant component reduced Mo(VI) to Mo(V), which led to the creation of a green phosphate/Mo(V) complex at an acid pH. This was the basis for the phosphor-molybdenum technique. Essentially, it is thought that the complex facilitates molybdenum reduction more easily, and that reductants and Mo(VI) undergo an electron-transfer reaction that results in the production of a green phosphate/Mo(V) complex with a maximum absorption at 695 nm.

Data analysis:

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RESULT AND DISCUSSION:

Phytochemical screening:

Preliminary phytochemical screening of the methanol extract of leaf of Annona reticulate Linn revealed the presence of different kind of chemical groups that are summarized in table 4.

Table 4. Result of chemical group test of the methanolic extract of leaves of Annona reticulata Linn

Key: MEAR-Methanolic Extract of Annona reticulata Linn.

[+Presence, -Absence]

There is evidence that the leaves of Annona reticulata Linn. contain a variety of phytoconstituents, including monosaccharides, alkaloids, glycosides, steroids, tannins, and flavonoids [14]. We found no combined lowering sugar. The extract's diverse pharmacological actions may be explained by these phytoconstituents.

Total Phenol Content Determination:

Using the Folin-Ciocalteu reagent, the total phenolic content of the methanolic leaf extract of Annona reticulata Linn was assessed and was expressed as Gallic acid equivalents (GAE) per gram of plant extract.The total phenolic contents of the test fractions were calculated using the standard curve of Gallic acid (Figure 2).

The methanolic extract of A. reticulata leaf was used to determine the total flavonoid content using the aluminum chloride colorimetric technique. The total flavonoid content was calculated (Figure 3) and was expressed as quercetin equivalents (QE) per gram of the plant extract. The methanolic extract of the leaf of A. reticulata was found to contain appreciable amount of flavonoids in this result.  The scavenging of free radicals, chelation of metal ions like iron and copper, and inhibition of free radical-generating enzymes are only a few of the methods by which flavonoids exhibit antioxidative characteristics [15].

Total Antioxidant Capacity:

Figure 4. Calibration Curve Of Ascorbic Acid

Brine Shrimp Lethality Bioassay for Cytotoxic Activity:

The extract was also subjected to Brine Shrimp lethality bioassay for possible cytotoxic action. In this study, methanol extract of leaf was found to be the most toxic to Brine Shrimp nauplii, with LC5o of 39.67 µg/ml whereas anticancer drug vincristine sulphate showed LCs value 0.699 µg/ml (table 5). The high toxicity of methanolic extract of leaf probably attributed to the alkaloid that is confirmed in phytochemical screening.