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INDEX – GJRMI - Volume 7, Issue 3, March 2018 MEDICINAL PLANTS RESEARCH Biological Sciences IN VITRO ANTIOXIDANT ACTIVITY OF METHANOLIC LEAF EXTRACTS OF TRIDAX PROCUMEBENS L., LANTANA CAMARA L. & TITHONIA DIVERSIFOLIA (Hemsl) A. Gray. FOUND IN PARTS OF NIGER- DELTA, NIGERIA Sam S M*, Jacobs I E, Johnny I I, Udosen I R, Mensah S I
COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – INFLORESCENCE OF WOODFORDIA FRUTICOSA (L.) KURZ.* OF THE FAMILY LYTHRACEAE PLACE – KOPPA, CHIKKAMAGALUR DISTRICT, KARNATAKA, INDIA *BOTANICAL NAME VALIDATED FROM www.theplantlist.org AS ON 30/03/2018
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Global J Res. Med. Plants & Indigen. Med. | Volume 7, Issue 3 | March 2018 | 27–33 ISSN 2277-4289│ www.gjrmi.com │International, Peer reviewed, Open access, Monthly online Journal
Short Communication IN VITRO ANTIOXIDANT ACTIVITY OF METHANOLIC LEAF EXTRACTS OF TRIDAX PROCUMEBENS L., LANTANA CAMARA L. & TITHONIA DIVERSIFOLIA (Hemsl) A. Gray. FOUND IN PARTS OF NIGER- DELTA, NIGERIA Sam S M1*, Jacobs I E2 Johnny I I3, Udosen I R4, Mensah S I4 1
Department of Biological Sciences, Akwa Ibom State University, Ikot Akpaden, Nigeria Department of Pharmacognosy and Natural Medicine, University of Uyo, Nigeria 4 Department of Biology, Akwa Ibom State College of Education, Afaha Nsit, Nigeria 5 Department of Plant Science & Biotechnology, University of Port Harcourt, Nigeria *Corresponding Author: Email: sundaysam@aksu.edu.ng, eosamviek@yahoo.com 2,3
Received: 05/02/2018; Revised: 21/03/2018; Accepted: 30/03/2018
ABSTRACT The aerial parts of three traditional medicinal plants Tridax procumbens, Lantana camara and Tithonia diversifolia were used to analyze the presence of bioactive secondary metabolites and to determine its ability to scavenged 2,2 diphenyl-picrylhydrazyl (DPPH) radical, Nitric Oxide radical (NO) and Hydrogen peroxide radical (H2O2). Different concentrations of methanolic extract were subjected to antioxidant assay by DPPH assay, Nitric Oxide scavenging activity, and hydrogen peroxide. The results showed that Tridax procumbens at a maximum concentration of 0.4mg/ml showed the scavenging percentage antioxidant activity of 57.2% followed by Lantana camara at 0.2 mg/ml which showed 47.4% and Tithonia diversifolia at 1.0 mg/ml showed 37.5% which was observed to be higher than those of abscorbic acid at 0.6 mg/ml which showed 17% used as standards. The extract of Tithonia diversifolia showed 83.0% at 0.2mg/ml, Tridax procumbens 23.0% at 0.2 mg/ml, Lantana camara 39.2% at 0.4 mg/ml of Nitric Oxide. The results therefore revealed that the three medicinal plants methanolic leaf extracts have significant (P<0.05) antioxidants which could serve as an easily accessible item of natural rich source of antioxidant, and also serve as a promising pharmaceutical. This suggests that the leaves of Tridax procumbens, Lantana camara and Tithonia diversifolia could be a good source of antioxidants to ameliorate conditions in diseases whose pathogenesis implicates oxidative stress. KEYWORDS: In vitro, antioxidants, scavenging, methanolic extracts Cite this article: Sam S M*, Jacobs I E Johnny I I, Udosen I R, Mensah S I (2018), IN VITRO ANTIOXIDANT ACTIVITY OF METHANOLIC LEAF EXTRACTS OF TRIDAX PROCUMEBENS L., LANTANA CAMARA L. & TITHONIA DIVERSIFOLIA (Hemsl) A. Gray. FOUND IN PARTS OF NIGERDELTA, NIGERIA, Global J Res. Med. Plants & Indigen. Med., Volume 7 (3): 27–33
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INTRODUCTION Nowadays many plants species have being discovered and used for their medicinal properties, Ethnobotany have become very useful to modern pharmacy, most of these plants and herbs, fruits, vegetables. Shashi and Rabinaranyan (2017) have reported the ethnomedicinal claims of Euphorbia caducifolia Haine which is a source of drug. Synthetic antioxidants have become toxic to human health and that’s why natural antioxidant, such as herbs, fruits, vegetables have gained interest among people in different parts of the world, studies have shown that plants intake as natural antioxidants is associated with low risk or no risk to cardiovascular diseases (Rimando and Perkins-Veazie, 2005). The protective effect in herbs and fruits are related to 3 groups these are phenolics, vitamins, carotenoids. Antioxidants are any substance that delay or inhibits oxidative damage to a target molecule, a single antioxidant molecule can react with single free radicals and are capable of neutralizing them by donating one of their own electrons, antioxidants prevent cell and tissue damage as they act as scavengers, and they protect the cells and organs and system against reactive oxygen species. Antioxidants are able to remove free radicals and prevent them from causing damage, these free radicals are responsible for a whole lot of health problems such as cancer, aging, heart diseases, and gastric problems. Basically antioxidant is any substance, when present in low concentration, significantly delays or slow oxidation of all contents, like lipids, protein, carbohydrates (Eze, 2006; Catala, 2009). Plants serve lots of purpose in human health today; studies have shown that phytonutrients gotten from fruits and herbs play preventive role in development of some diseases (Diplock et al., 1998). It’s widely accepted that natural antioxidants found in herbs and fruits can fight against human disease, reactive oxygen species, nitrogen species (Halliwell, 1997). Studies have shown that high intake of plants can reduce chances of cancer (Doll et al., 1990). Herbs, fruits, vegetables with high phenolic content are important in human dietary and ethnobotany
providing protection against cellular damage caused by exposures to high levels of free radicals. Plants are made up of several antioxidant compounds that serve as radical scavengers. Tridax procumbens L. belongs to the family of Asteraceae commonly known as coat button. It is a spreading annual herb grows up to 20 cm in height, it has simple opposite leaves serrate or dentate, acute shape, fleshy pubescent, flowers in head, long stalk, with white seeds, are numerous, small with tuft and silky hairs, its seen as noxious weed and pest plant and has been valued for its medicinal properties (Sahoo and Chand, 1998). Lantana camara L. is a flowering plant in the family of Verbenaceae, commonly known as wild red sage. It is a gregarious erect half climbing and aromatic shrub, grows up to 1.2 m in height and branches all four side with recurved prickles leaves are elliptic about 3inch long, opposite. It is being listed as one of the important medicinal plant in the world (Ross, 1999). All parts have been used traditionally in treating ailments due to its multiple levels of biological activities (Patel et al., 2011). Tithonia diversifolia (Hemsl.) A. Gray belongs to the family of Asteraceae commonly known as Mexican sunflower. In Nigeria, the decoctions of various parts of the plant are used for the treatment of malaria, diabetes, sow throat and menstrual pains (Elufioye, 2004, Owoyele, 2004). Oxidative stress is linked to information, associated with pathogenesis cancer (Maeda and Omata, 2008), Cardiovascular diseases (Montecco, 2011). Oxidative stress occur when there is an inhibition between antioxidants defense and ROS (Betteridge, 2000). The redox stress triggers the activation of immune cells which releases pro inflammatory, the oxidative stress as being termed to shift toward oxidants. In biological systems, reactive oxygen species are continuously generated and they play a role in the pathogenesis of many disease conditions by causing extensive damage to biomolecules and tissues (Eze, 2006; Catala, 2009). Oxidative
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stress is associated with heart diseases and is a harmful condition that occurs when there is an excess of ROS or a decrease in antioxidants levels it leads to damage by physical, chemical, psychological factors that leads to tissue injury in human and develop different diseases. These free radicals generally are molecules present in the environment and also occur in body as natural part of physiological function, they cause cell damage, however and when in high levels may contribute to chronic conditions. It is impossible to avoid free radicals, but antioxidant can minimize all these effects, a rich source of antioxidants is through vegetables, fruits and herbs.
Preparation of extracts
This study therefore was aimed to investigate antioxidant activities variation in three medicinal plants commonly found in Niger Delta region of Nigeria using different invitro models.
Antioxidant ability assays
MATERIALS AND METHODS Chemicals Methanol, 1,1,diphenyl 2 picryhydraxy (DPPH), Hydrogen peroxide, Sodium Nitroporoxide, Sulfanilic acid, Ascorbic acid, Naplthylene Diamine Dichloride, Acetic acid, Sulfuric acid, Ammonium molybdate. All chemicals were purchased from Jeochem Nigeria limited. Collection of plant materials The plants Tridax procumbens L., Lantana camara L. and Tithonia diversifolia (Hemsl.) A. Gray were collected from the environs of Akwa Ibom State University, Ikot Akpaden and were identified by a taxonomist in the Department of Biological Sciences, Akwa Ibom State University and voucher specimen was deposited in the Akwa Ibom State University Herbarium (AKSU) for reference purposes. The plants collected were air dried and pulverized into coarse powder.
The air dried and pulverized plant materials weighing 200g each were soaked in methanol 100% for 2 days with intermittent shaking, the solvent was filtered using a whattman filter paper, the filtrate was evaporated to dryness using an oven and dark green mass residue was obtained and stored in 4°C till further use. Phytochemical analysis Preliminary standard laboratory measures were used to test for various phytochemicals of individual plant extract.
This was evaluated using phosphomolydenum method with the procedure of (Prito et al., 1999). 0.3ml of various plant extracts was combined with 3 ml of reagent solution (0.6mM sulfuric acid, 28mM sodium phosphate, 4Mm ammonium molybdate). The test tubes were incubated at 95°C for 1.30 mins thereafter absorbance was measured, ascorbic acid serve as standard. DPPH radical scavenging assay The free radical scavenging activity of the extract was measured using DPPH by method of (Gyamfi et al, 1999) with slight modifications by Awah et al, (1989). 1mM solution of DPPH in methanol was added to 3ml of various concentration of plant extract ranging from (0.2–1.0mg/ml) of sample dissolved in methanol and kept in dark room for 60mins, absorbance was measured at 517nm; ascorbic acid was used as control. Nitric oxide scavenging assay The method of Marcocci et al, (1994) was used with slight modifications by Awah et al, (1989). The extract of individual plants were mixed with sodium Nitroporoxide. 10mM in phosphate buffer saline (pbs) incubated at 25°C for 150mins, 0.5ml of griess reagent was added to each test tube (0.33% Sulfanilic acid prepared in 20% glacial acetic acid) at room temperatures
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for 5min and 1ml of Napthelene Diamine Dihydrocloride (0.1%w/v). The mixtures were incubated at room temperature for 30 mins, measured in UV spectrometer; ascorbic acid served as control.
Statistical analysis
Hydrogen peroxide scavenging assay
RESULTS
Hydrogen peroxide scavenging potential of the individual extract was determined using the method of Jajaprakachi et al. (2004). A solution of hydrogen peroxide 20mM was prepared in phosphate buffer saline (7.4). 2ml of the solution was added to different concentrations of the extract ranging from 0.2 to 1.0 ml/mg, and incubated at room temperature for 10mins, absorbance was measured and ascorbic acid served as control.
Phytochemical analysis : The results of the phytochemical analysis revealed that the three plants extracts contains terpenes, flavonoids, phenols, glycosides and alkaloids while tannins and saponin were absent in Tridax procumbens (Table 1).
All analysis were replicated tries, and data were expressed as mean+/- SD analysis variance.
Total antioxidant activity: The result of the analysis showed that Tithonia diversifolia has maximum absorbance of 1.137 at 695nm, while Tridax procumbens has 0.955, and Lantana camara 0.710 (Figure 1).
Table 1: Results of Preliminary Phytochemical Analysis Phytochemical constituents Terpenes Flavones Saponin Tannins Glycosides Phenols Alkaloids
Tridax procumbens
Lantana camara
+ + + + + + + + + + + + KEY: + = present - = Absent Figure 1: Antioxidant capability assay
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Tithonia diversifolia + + + + + +
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Free radical scavenging activity: The result of DPPH radical scavenging activity showed that Tridax procumbens has more antioxidant power 57.2% at 0.4mg/ml while Lantana camara has more capacity at 0.2mg/m showed 47.4%, Tithonia diversifolia at 1.0 mg/ml showed 37.5% (Figure 2). Nitric Oxide scavenging activity: The result of NO scavenging showed that Tithonia diversifolia has a maximum absorbance of 83.0% at 0.2 mg/ml, while that of Tridax procumbens was 23.0% at 0.2mg/ml and Lantana camara 39.2% at 0.4 mg/ml, the
abscorbic acid as standards showed higher NO scavenging when compared to the extracts (Figure 3). Hydrogen peroxide: In the Hydrgen peroxide, Tithonia diversifolia showed more maximum absorbance at 0.8mg/ml ie. 2.954 whereas Tridax procumbens showed maximum absorbance at 1.0mg/ml ie. 2.234, and Lantana camara showed maximum absorbance at 0.4mg/ml ie. 2.822 (Figure 4).
Figure 2: DPPH radical scavenging activity.
Figure 3: Nitric Oxide scavenging activity
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Figure 4: Hydrogen peroxide scavenging activity
DISCUSSION Medicinal plants have become extremely popular all over the world as antioxidants and in recent past, several dietary and herbal formulations, which have free radical scavenging potential are relevant in the treatment of chronic diseases (Tiwari and Tripathi, 2007). In biological systems, reactive oxygen species are continuously generated and they play a role in the pathogenesis of many diseased conditions by causing extensive damage to biomolecules and tissues (Eze, 2006; Catala, 2009). In figure 1, there is a positive correlation between phenolic content and total antioxidant activity of extracts. Tithonia diversifolia showed a maximum absorbance at 1.137, while Tridax procumbens has 0.955, and Lantana camara has 0.710. The extract showed a potent DPPH radical scavenging potential. Tridax procumbens showed more DPPH scavenging activity of 57% at 0.4mg/ml than Lantana camara and Tithonia diversifolia. Free radicals are chemical entities that can exist separately with one or more unpaired electrons. The change in absorbance of DPPH radical caused by antioxidants is due to the reaction between the antioxidant molecules and the radical, which results in the scavenging of the radical by hydrogen donation Naik et al., 2003). In figure 3, Tithonia diversifolia has a maximum absorbance of 83% at 0.2mg/ml than Tridax
procumbens and Lantana camara. Nitric oxide is regarded as an important mediator of acute and chronic inflammation, which can easily react with superoxide anion to form peroxynitrite (ONOO-), a potent oxidizing molecule capable of eliciting lipid peroxidation and cellular damage (Beckman et al., 1990; Radi et al., 1991; Rubbo et al., 1994). Figure 4 showed that Tithonia diversifolia shows maximum absorbance at 0.8mg/ml 2.954 than Tridax procumbens and Lantana camara. Among the major active oxygen species the hydroxyl radical (OH¡) is the most reactive and severely damages adjacent macromolecules such as proteins, lipids, nucleic acids and almost every macromolecule in vicinity. Therefore, the removal of hydroxyl radical is probably one of the most effective defenses of living body against various diseases. CONCLUSION This study shows that the methanolic leaf extracts of Tridax procumbens, Lantana camara and Tithonia diversifolia exhibited significant antioxidant activity in the different in vitro free radicals. This may be related to the presence of flavonoids, phenols and tannin compounds in the plant extracts. The plant extracts could be useful in preventing and slowing the progress of various oxidative stress induced disease.
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REFERENCES Awah, F. M, Offor, N. N, Ndunaka, A. C, Okafor, F. U, Enyabine, C. O. (2012). Free radical scavenging activities and phenolic contents of the spices Thymus vulgaris (Thyme), Helichrysum italicum (Curry leaf) and Laurus nobilis (Bay leaf) Extracts. J Pharm Res. 5(6); p2994 Betteridge D.J: (2000). "What is oxidative stress?". Metab. 49(2):3–8 Catala,A. (2009). Lipid peroxidation of membrane phospholipids generates hydroxy-alkanals and oxidized Phospholipids active in physiological and/or pathological conditions. Chem. Phys. Lipids.157 (1):1–11. Eze, M. O. (2006). The oxygen paradox and the place of oxygen in our understanding of life, aging, and death. Ultim.Real Meaning (URAM):stud.Med.Health.29 (1):46–61. Gyamfi, M. A, Yonamine, M, Aniya, Y. (1999). Free-radical scavenging action of medicinal herbs from Ghana: Thonningia sanguine on experimentallyinduced liver injuries. Gen. Pharmacol. 32:661–667. Halliwell, B. (1997): Antioxidants and human diseases: a general introduction, Nutrition Reviews, 55, 44–46. Maeda, S; Omata, M. (2008): "Inflammation and cancer: role of nuclear factor-kappa B activation." Cancer Sci. 99(5):836– 842.
Source of Support: NIL
Marcocci, P. L, Sckaki, A, Albert, G. M. (1994). Antioxidant action of Ginkgo biloba extracts EGP761. Methods Enzymol. 234:462–475. Montecco, F; Pende A, Quercioli A, Mach F. (2011). "Inflammation in the pathophysiology of essential hypertension." J. Nephrol. 24(1):23–36 Naik G. H., Priyadarsini K. I., Satav J. G., Biyani, M. K. and Mohan, H. (2003). pathological conditions. Chem. Phys. Lipids.157 (1):1–11. Rainard, R, Prawez S, Verma, P.K and Pankaj, N. (2008): Medicinal Plants and their Role in Wound Healing, Vet Scan, 3(1), 221–224. Sahoo, M. and Chand, P. K, (1998); Invitro multiplication of a medicinal herb Tridax procumbens L. (Mexican Daisy, coat button): influence of explanting season, growth regulator synergy, culture passage and planting 6(23):4032–4038. Shashi Gupta, Rabinarayan Acharya (2017), An Appraisal On Ethno-Medicinal Claims Of Euphorbia Caducifolia Haines. – A Source Drug Of Ayurvedic Medicinal Plant ‘Rakta-Snuhi’, Global J Res. Med. Plants & Indigen. Med., Volume 6 (3): 54–63 Tiwari,
A.K. (2004): Antioxidants newgeneration therapeutic base for treatment of polygenic disorders, Current Science, 86, 2004, 1092–1102.
Conflict of Interest: None Declared
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