ANTIOXIDANT ACTIVITY TOTAL PHENOLIC AND FLAVONOID CONTENTS OF WHOLE PLANT METHANOL EXTRACT AND SOLVENT FRACTIONS OF DESMODIUMRAMOSISSIMUM G. DON

ABSTRACT

The present study was designed to estimate the total phenolic and flavonoid contents and to evaluate the in vitro antioxidant activities of both the methanol extract and the various solvent fractions of the whole plant, Desmodiumramosissimum. Qualitative phytochemical screening showed the presence of various bioactive components such as phenolics, flavonoids and tannins. The quantitative analyses of total phenolic content (TPC) and total flavonoid content (TFC) were carried out spectrophotometrically using Folin-Ciocalteu phenol reagent and aluminum chloride method, respectively. Ethyl acetate fraction followed by n-butanol fraction exhibited high total antioxidant activity, DPPH radical scavenging ability as well as ferric ion reducing ability when compared to all other solvent fractions including the methanol extract. The presence of these flavonoid and phenolic compounds might contribute to the antioxidant properties observed in these fractions. In order to support this observation, the relationship between the antioxidant content using various models revealed that TPC exhibited a strong correlation with ferric ion reducing potential (FIRP) and total antioxidant capacity (TAC) with R2  values of 0.995 and 0.980, respectively; but a weak relationship with 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical with R2 value of 0.877. However, aqueous fraction showed a strong relationship between DPPH and its concentration compared to other fractions. This showed that various solvent extracts possess unique chemical constituent of plant with various antioxidant capacity. Free radicals were scavenged by the extract and solvent fractions in a concentration-dependent manner within the range ofthe given concentrations (25-1000 mg/l) in all models. The findings from this study show that  methanol extract  and  the  solvent  fractions  of Desmodiumramosissimum G.  Don contained phenolics, flavonoids and tannins which exhibited antioxidant properties.

CHAPTER ONE

INTRODUCTION

Since the last decade, the concept of health promotion has become a legitimate part of health care and as such, there is a growing interest in natural antioxidants as against the synthetic counterpart  because  of  the  concern  over  their  features  such  as  their  volatile  nature,  high sensitivity to heat and instability with possible carcinogenic effects (Kurian et al., 2010; Ruiz- Navajas et al., 2011). The intake of natural antioxidants has been associated with reduced risks of cardiovascular disease, cancer, diabetes, and diseases associated with ageing (Hamid et al.,

2010). Therefore, antioxidants have  become  very well recognized nutraceutical ingredients. Plants, particularly, medicinal plants are of great importance to the health of individuals and communities. Many people in the world have difficulty in gaining access to modern medicine and as such, they use traditional medicine which is based on the fact that medicinal herbs and plants are used as an alternative to conventional treatment for their recovery (Muanda et al.,

2011).

Plants have been known to be good and rich sources of potential bioactive compounds which are of high benefit  to  health (Doughari, 2012). These bioactive chemical compounds are often referred to as ‘phytochemicals’. There are various mechanisms of action of phytochemicals. Phytochemicals may either be used as chemotherapeutic or chemopreventive agents. According to Kotzekidou et al. (2008), the mechanism of action of phytochemicals is considered to be the disturbance of the cytoplasmic membrane, disruption of the proton motive force, disruption of the electron flow and active transport, as well as the coagulation of cell contents. In addition to these, plant extract can inhibit  microorganisms by interfering with some of their metabolic processes, modulate gene expression and signal transduction (Surh, 2003).

Williams et al. (2004) hypothesized that cells respond to phytochemicals through direct interactions with receptors or enzymes involved in signal transduction, or through gene expression modification that may result in the alteration of redox status of the cell that could trigger a series of redox-dependent reactions. There is evidence that flavonoids may play an important role in molecular processes especially as modulators of intracellular signaling cascades

which are vital for cellular functions. The abilities of phenolic-based antioxidant therapies in decreasing the reactive oxygen species (ROS) levels have been shown to produce the best health benefits through a diet  rich in  multiple antioxidants rather than a  high dosage of a  single supplement (Lee and Lee, 2006).

With an increase in civilization and technology, man is now constantly and increasingly being exposed to the pollution of his natural environment (such as smoking) as well as alcohol abuse, improper nutrition, living a stress-bearing and non-hygienic lifestyle. All these pose serious risk to health loss. Oxidative stress, which results from the imbalance between reactive oxygen species (ROS) and the biological system’s ability to detoxify the reactive intermediates, appears to be the main cause of many diseases such as Parkinson’s disease, ulcer (Atawodi, 2005), premature aging of the skin (Jayasri et al., 2009). Hence, oxidative stress results as these excess ROS cause damage to biological molecules leading to disease conditions (Uddin et al., 2008). Any substance or molecule of antagonist effect with respect to the production of free radicals is known as an antioxidant. Studies by D’abrosca et al. (2007) and Tsao (2010) revealed that flavonoids have a strong antioxidant potential. In addition to this finding, earlier studies by Mattson and Cheng (2006), and Yusufoghu and Alqusoumi (2011) showed that phenolic compounds and flavonoids protect cell constituents through direct scavenging of free radicals due to their antioxidant properties. However, recent data indicated that the protective effect of flavonoids and phenolic compounds may extend beyond their antioxidant activity (Jaganath and Crozier, 2010; 2011).

The traditional medicinal use of Desmodium ramosissimum lies in the fact that the herbs of this genus, Desmodium, possess wide and potent pharmacological properties that can form a practical base for further scientific research. For instance, D. ramosissimum is used in the treatment of dysentery, eye diseases and fever, and as excitant in some parts of Africa (Uphof, 1968). It is also medicinally used in West Tropical Africa, Madagascar and La Reunion (Caius, 1989) for the treatment of fever. It is, however, necessary to find out whether there are available pharmacological compounds in this specie of Desmodium to validate their traditional use as medicine.

1.1      Plant and medicine

Plants are naturally endowed with inexhaustible and invaluable active ingredients used in the management of many intractable diseases. Phytochemical analysis has played a significant role in  searching  for  raw  materials  and  resources  for  the  pharmaceutical industry.  Preliminary phytochemical tests are helpful in the search and identification of active chemical constituents which are sources of pharmacologically active principles (Pandith, 2012).

Fabricant  and  Farnsworth (2001)  reported that  about  80  percent  of the  active  compounds discovered in plants show a positive correlation between their modern therapeutic applications and the traditional uses of plants from which they are derived. The concept of drug synergism is also used in pharmaceutical research. However, clinical trials should be used to investigate the efficacy of a particular herbal preparation, provided the formulation of that herb is consistent (Izhaki, 2002). The knowledge of the chemical constituents in plants is desirable because such information may be of value in disclosing new sources of such chemical substances and it can be possible also that these chemical constituents of plants may be discovered to be of therapeutic importance (Sahoo et al., 2013).

1.1.1    Desmodium Species

The genus Desmodium belongs to the Tribe of Desmodieae and to the family, Leguminosae (also known as Papilionoideae) which is extensively distributed in the tropic, sub-tropic and temperate regions of the world-Africa, Madagascar, Southeastern and Eastern Asia,  Australia and the American Continent (Adriano et al., 2010). About 350 species have been found distributed in tropical and subtropical regions of the world while 28 species have been found in China (Liguo et al., 2008). Majority of Desmodium plants are herbs, shrubs or sub-shrubs but are rarely found as trees. Besides their popularity as forage, they are also used in traditional medicine.

Ma et al. (2011) reported that the use of Desmodium species for ethnomedicinal purposes in China dates back to as early as 3000 years ago. In China, over twenty (20) species in this genus have a long history of medical use in Traditional Chinese Medicine (TCM). According to the theory of Traditional Chinese Medicine (TCM), the herbs of this genus, Desmodium, have been mainly used to relieve internal heat or fever, neutralize toxins, inhibit pain, invigorate blood

circulation, suppress cough and alleviate dyspnea (Editorial Committee of Jiangsu New Medical College, 1977; Editorial Committee of Zhonghua Bencao, 1996; Editorial Committee of Quan Guo Zhong Cao Yao, 1996; Editorial Committee of Chinese Materia Medica, 1999). These herbs are also found abundant in India and have also been extensively used as traditional medicines in Ayurveda to treat various diseases (Rastogi et al., 2011).

1.1.2   Mode of preparation and Use

In the preparation of Desmodium plant for its medicinal value, the whole plant is commonly used, however, the aerial parts such as the leaves and stems can also be used. The plant may be administered orally  or  applied  topically,  in  the  case  of treating  skin  ailment  or  bite.  The extraction of the chemical constituents of these plants may include boiling the plant parts in water (decoction), chewing the fresh leaf (as in treating dysentery and diarrhea), and grinding the fresh leaf to extract the juice. In some rare cases, the leaves can serve as vegetable used to garnish or steam foods but mainly for the purpose of treating illness rather than its nutritive or aromatic value (Editorial Committee of Guizhou Institute of Chinese Medicine, 1970).

Furthermore, it is now understood through in vivo and in vitro experiments that various Desmodium plant extracts possess a wide range of pharmacological properties which include bioactive substances involved in improving cardiovascular and cerebrovascular functions, regulating immune system as well as anti-inflammatory, cytotoxic, anti-parasitic, anti-diabetic, anti-nephrolithic, antibacterial, and nootropic activities (Ning et al., 2009; Zhu et al., 2010). It has been reported also through various researches that flavonoids and alkaloids are the principal constituents and perhaps, are responsible for most of the medicinal activities shown by the plants of this genus (Gan et al., 2008).

This versatile medicinal traditional uses of Desmodium plant have aroused an increasing number of phytochemical studies on this plant genus. In this respect, over 200 compounds including flavonoids, alkaloids, steroids, terpenoids, phenylpropanoids and other constituents have been isolated from the various species of this genus, however, only a few have been evaluated for their biological activity (Rastogi et al., 2011).

1.1.3   Medicinal/Pharmacological properties of Desmodium plants

A brief review of various species of Desmodium revealed that this genus has huge medicinal potentials as seen in their pharmacological properties which include:

1.1.3.1 Anti-inflammatory and anti-pyretic

The petroleum ether fraction (PEF) from the ethanol extracts of Desmodium podocarpum, at an oral dose possesses noticeable activity against acute inflammation in mice caused by dimethylbenzene (Zhu et al., 2010). In addition, the oral administration of PEF of Desmodium podocarpum significantly decreased lipopolysaccharide (LPS)-induced fever in rats in a time and dose-dependent manner (Zhu et al., 2010). Gangetin, a pterocarpenoid isolated from Desmodium gangeticum, exhibited potent anti-inflammatory property (Ghosh and Anandakumar, 1983).

1.1.3.2 Analgesic activity

The water decoction of roots and aerial parts of Desmodium gangeticum were proved to have analgesic activity (Rathi et al., 2004).

1.1.3.3 Antioxidant activity

The methanol-water extract of Desmodium adscendens leaves was shown to possess a considerable scavenging antioxidant activity (Muanda et al., 2011). The methanol extracts of Desmodium triflorum also exhibited potent antioxidant activities (Mao et al., 2007). Also, the pretreatment of rats with Desmodium triflorum extract significantly (p < 0.001) increased the activities of superoxide dismutase, glutathione peroxidase and glutathione reductase induced by ʎ-carrageenan (Lai et al., 2009). Ethyl acetate fraction of Desmodium triflorum was the most active in scavenging DPPH and trolox equivalent antioxidant capacity (TEAC) radicals (Lai et al., 2010). Desmodium gangeticum also demonstrated a potent DPPH scavenging activity regardless of the methods used in its preparation (Rastogi et al., 2011) while 95% alcohol extract of Desmodium styracifolium  evaluated  by photometric  method  revealed  a  high  scavenging percentage of this extract against hydroxyl, superoxide and free radicals generated from cigarette smoke. Desmodium styracifolium alcohol extract also exhibited inhibitory effect on lipid peroxidation (Mao et al., 2007).

1.1.3.4 Cardio-protective activity

The  ethyl  acetate  fraction  of  the  alcohol  extract  of  Desmodium  gangeticum  exhibited  a significant improvement of cardiac function and a reduction in the release of lactate dehydrogenase in coronary effluent, as well as a reduction in the level of malondialdehyde in myocardial tissues (Kurian et al., 2010).

1.1.3.5 Antimicrobial and antiparasitic activity

Ethanol extract of Desmodium caudatum roots showed antimicrobial activity in vitro against Bacillus subtilis, Staphylococcus aureus, Mycobacterium smegmatis, and Streptococcus facialis (Delle-Monache  et  al.,  1996).  The  aqueous,  alcohol  and  acetone  extracts  of  Desmodium barbatum also  exhibited  significant  antibacterial activity (Jimenez-Misas et  al.,  1979).  The chloroform-methanol (1:1) extract derived from Desmodium grahami produced a concentration- dependent antimicrobial activity against pathogenic enterobacteria in vitro (Rojas et al., 1999). Plants of the genus Desmodium were used to protect cereals from being parasitized by Striga hermonthica, an obligate parasitic weed that can devastate the maize crop (Hooper et al., 2009,

2010; De Groote et al., 2010; Khan et al., 2010).

A novel flavonoid, isoschaftoside, isolated from the root exudate of Desmodium uncinatum demonstrated inhibitory effect on the haustorial development of Striga and stimulated suicidal germination of Striga hermonthica seeds, dramatically inhibiting its parasite in the host roots. Other plants of this  genus  like Desmodium pringlei, Desmodium introtum and Desmodium sandwicense also showed similar effects (Khan et al., 2002; Khan et al., 2006).

1.1.3.6 Antidiabetic activity

Alcohol extracts of Desmodium gangeticum aerial parts was reported to possess both in vitro and in vivo anti-diabetic properties. In addition, the HPLC analysis of the extract revealed that the significant anti-diabetic and lipid lowering activity of Desmodium gangeticum may be attributed partially to the presence of chlorogenic acid (Govindarajan et al., 2007).

1.1.3.7 Antinepherotic activity

A terpenoid isolated from Desmodium styracifolium referred to as Soyasaponin I has been shown to be effective against urolithiasis. Its anti-nephrolithic effect was reported to be on the formation of calcium oxalate renal stones induced by ethylene glycol. The inhibition on the formation of calcium oxalate stones in kidneys by Soyasaponin I increased the output of urine, decreased the excretion of calcium and increased the urinary excretion of citrate (Kubo et al., 1989; Hirayama et al., 1993).

1.1.3.8 Hypotensive activity

The aqueous extract of Desmodium styracifolium exhibited potent hypotensive activity both in vivo and in vitro (Ho et al., 1989).

1.1.3.9 Anti-anaphylactic activity

The anti-anaphylactic properties of aqueous extract of Desmodium adscendens leaves and stems showed that the extract, given orally, caused a concentration-dependent reduction in the extent of the anaphylactic contraction in guinea pigs (Addy and Awumey, 1984; Addy and Dzandu, 1986).

1.1.3.10 Wound-healing activity

The ethanol extract of Desmodium triquetrum leaves, exhibited significant (p < 0.005) increase in the tensile strength after a wound in the incision wound model. In addition, this extract significantly enhanced epithelization and facilitated wound contraction (Shirwaikar et al., 2003).

1.1.3.11 Immunomodulatory effect

A sum of flavonoids from Desmodium canadense, significantly enhanced the phagocytic activity of neutrophils both in vivo and in vitro showing an increased number of phagocytied cells and phagocytose index (Kondrotas et al., 1997).

1.1.3.12 Anti-fibrotic activity

Aqueous and  alcohol extracts  of the  whole  plant  of Desmodium pulchellum  proved  to  be effective in inhibiting the liver damage induced by carbon tetrachloride (CCl4) in rats, and decreasing the hepatic fibrosis (Ming et al., 1999; Ming et al., 2001).

1.1.4 Desmodium ramosissimum

Figure 1: A photograph of Desmodium ramosissimum (Hyde et al., 2015)

1.1.4.1 Description of the plant Desmodium ramosissimum

D. ramosissimum is an erect, slender perennial herb of about 0.3-2 m in height. It stems severally from a woody base with fine short hairs and longer white hairs or sometimes densely covered with appressed stiff hairs. The leaves are arranged in three (3) foliolate with leaflets narrowly obovate to oblong-elliptic, hairless above and appressed hairy beneath with reticulate venation visible on both surfaces. Flowers are usually of standard mauve, red or rose-colour having fruit of about 7-25 mm long, and covered with short hooked hairs as shown in Figure 1 above (Hyde et al., 2015).

1.1.4.2 Scientific classification

Kingdom:       Plantae Order:             Fabales Tribe:              Desmodieae Family:           Leguminosae Genus:            Desmodium

Species: ramosissimum Subspecies: ramosissimum (Hyde et al., 2015)

Table 1: Common names of D. ramosissimum

Country	Tribes	Common name
Sierra Leone	Koranko Loko	Dσnde Bσtugwe
Tanzania	Mende Bukoba District	b’aŋba Kasikasiki
Nigeria	Hausa Igbo	waken zoomoo bean of the hare oganana
	Awka Enugu	Obin Egbarigba
	Ebira	Oweyi
(Burkill, 1985)

1.1.4.3 Ethnomedicinal uses of D. ramosissimum

Mainen et al. (2009) reported that in Bugabo Ward, Bukoba District of Tanzania, D. ramosissimum is called  Kasikasiki, and when the roots and  leaves of this plant  are boiled together with those of Tragia furialis and Clerodendrum buchanannii in water, a glass of this decoction administered daily increases libido while in Enugu State in Eastern Nigeria, as shown in Table 1, the plant is called ‘Egbarigba’. In Enugu State, the leaves are washed and chewed as a cure for dysentery. ‘Oweyi’, as it is called in Ebiraland of northern Nigeria, it is used to treat diarrhea, dysentery, fever, pulmonary troubles, cough, venereal diseases and jaundice (Alli et al.,

2011). Desmodium ramosissimum was also  reported to  be used for traditional treatment  of dysentery, eye diseases and fever in Bauchi state of Nigeria (Adamu et al., 2005). Cameroon (Owondo-bekon) and Southern Uganda usually use this plant for abortion. Two handfuls of leaves are macerated in 1 L of water, then, a cupful is drunk once a day for 2 or 3 days. This recipe was claimed to be effective when added with Basella alba or Bidens pilosa. It is also used for skin health, in which the decoction of whole plant of D. ramosissimum with leaves of Gloriosa superba and Momordica is used to bathe infants (Noumi and Tchakonang, 2001). Table

2 below shows the various uses of the different parts of D. ramosissimum.

Table 2: Various ailments treated with different parts of D. ramosissimum

Uses	Plant part	Ailment
Medicine	Leaf sap	Pain-killer
	Leaf sap Leaf	Eye treatment Ear treatment
	Leaf Leaf	Pulmonary troubles Diarrhea
	Leaf Leaf	Dysentery Menstrual cycle
Agriculture	Leaf Leaf	Venereal diseases Fodder
Industrial	Leaf Leaf	Dye Inks
Social	Leaf Leaf	Mordants Superstition
	Leaf	Magic

(Hyde et al., 2015)

1.2      Oxidative Stress

Mitra et al. (2014) defined oxidative stress as a disturbance in the pro-oxidant–antioxidant balance in favour of the pro-oxidant, leading to potential damage. This random, indiscriminate damage to a wide range of biomolecules is often called ‘oxidative damage’ (Halliwell and Whiteman, 2004). The pathogenesis of most diseases involves free radicals that mediate lipid peroxidation (Ogugua, 2000).

All aerobic organisms produce, at least, minimal levels of ROS, mostly arising from the side- production of superoxide ion during the reduction of molecular oxygen by mitochondria. Hydrogen peroxide (H2O2) produced by oxidases such as monoamine oxidase and NADPH oxidase can result in greater oxidative stress susceptibility in tissues enriched in these enzymes. Accumulating levels of diffusible nitric oxide (NO), a second messenger in neurotransmission and signaling molecule and superoxide (O2-) give rise to peroxynitrite. The term nitrosative stress results from peroxynitrite and related reactive nitrogen species (RNS) which are capable of both oxidation and nitration of the aromatic side-chains of tyrosine and tryptophan (Sayre et al., 2001). Hydroxyl radical (OH.) is usually generated by gamma radiation but commonly generated physiologically by Fenton reaction because reduced transition metals react indiscriminately with all bio-macromolecules (Sayre et al., 2001).

1.3      Phytophenol

Phytophenol is a term used to define the abundance of naturally available phenolic rings with or without substituent such as hydroxyl or methoxy group(s). Phytophenols are secondary metabolites of plants. Secondary metabolites, other than providing plants with unique survival or adaptive features, are of commercial significance to mankind. They have been used in industries as dyes, fibers, glues, oils, waxes, flavouring agents, drugs and perfumes. They can serve as potential sources of new natural drugs, antibiotics, photoprotectant, insecticides, and herbicides (Surget et al., 2015; Croteau et al., 2000; Dewick, 2002).

Plants (which include fruits, vegetables, medicinal herbs, etc.) contain a wide variety of free radical-scavenging molecules, such as phenolic compounds (e.g., phenolic acids, flavonoids, anthocyanins, and tannins), nitrogen compounds (alkaloids, amines, and betalains), vitamins, terpenoids (including  carotenoids),  and  some  other  endogenous  metabolites,  which  possess antioxidant  activity  (Shekhar  et  al.,  2011;  Noori,  2012).  These  phytophenolic  compounds provide protection against ultraviolet radiation, pathogens, and herbivores in plant. The anthocyanin co-pigments in flowers attract pollinating insects and are responsible for the characteristic red and blue colours of berries, wines, and certain vegetables (Wiczkowski and Pisku, 2004).

₦5,000.00 – DOWNLOAD FULL PROJECT DOWNLOAD FULL PROJECT Added to cart

---

---

---

---

Related Project Topics :