The present thesis entitled "Angiotensin converting enzyme inhibitory and anti-oxidant activities of seeds and leaves of Raphanus sativus L. and seeds of Apium graveolens L. using in vitro models" deals with the exploration of pharmacological and phytochemical screening of the selected Indian medicinal plants, which are traditionally used by the local people and tribals in India to treat
various disease conditions like asthma, indigestion, bronchitis, cough, renal disorders, etc., and used as an antispasmodic, diuretic, antiscorbutic, astringent, digestive, etc (Krithikar and Basu, 1987).
The extracts screened for its phyto-chemical constituents revealed the presence of flavonoids and phenolics, accounting for its antioxidant property. Flavonoids are a group of polyphenolic compounds which possess numerous biological effects such as anti-inflammatory, anti-hepatotoxic, anti-ulcer activities, etc. They are also found to inhibit ACE ((Loizzo et al, 2007).
The biological mechanisms by which flavonoids modulate vascular function and blood pressure appear to be associated with the action of nitric oxide. Although the primary events leading to an increased production of NO• are not totally identified, it is generally accepted that the regulation of the renin angiotensin system in endothelial cells could play an important role in the control of NO• production. ACE, a crucial enzyme in the regulation of the renin angiotensin system, is a zinc-containing peptidyl dipeptide hydrolase whose active site consists of 3 parts: a carboxylate binding moiety such as the guanidinium group of arginine, a pocket that accommodates a lipophilic side chain of amino acid residues at C-terminal and a Zn ion. The Zn ion binds to the carbonyl group of the penultimate peptide bond of the substrate, thereby making the carbonyl group polarized and subjecting it to a nucleophilic attack. Therefore, some flavonoids were suggested to show in vitro activity via the generation of chelate complexes within the active centre of ACE, and also by the formation of hydrogen bridges between the inhibitor and amino acids near the active site (Ojeda et al., 2010). Free hydroxyl groups of phenolic compounds are also considered to be important structural moieties to chelate the zinc ions, thus inactivating the ACE activity (Loizzo et al, 2007).
High blood pressure is mostly associated with coronary heart diseases, end-organ diseases (e.g. retinopathy, renal failure) and increasing risk of stroke. In some patients with mild hypertension, a reduction in body weight and salt intake may be sufficient, but in majority of the cases drug treatment is essential. The β-adrenoceptor antagonists and thiazide diuretics are presently the first line of drugs used in the treatment of hypertension but several groups of drugs, by different mechanisms, reduce blood pressure by decreasing vasoconstrictor tone and hence peripheral resistance. The most important of these are the ACE inhibitors. The membrane bound ACE facilitates the rapid conversion of inactive peptide angiotensin I to active peptide angiotensin II by the cleavage of dipeptide units from the former substrate. Angiotensin II is a potent vasoconstrictor and has both direct and indirect action on the blood vessels which results in increased total peripheral resistance thus accounting for high blood pressure. (Neil, 2002)
The ACE inhibitors hinder the conversion of angiotensin I to angiotensin II by inhibition of the enzyme. In general, ACE inhibitors are widely used in combination with a thiazide diuretic when single drug therapy fails. However it has been observed that ACE inhibitors induce side effects such as dry cough and angioneurotic edema, skin rash, dysgeusia and neutropenia (Hoffman, 2002). Thus, new alternatives from plant source with an increased therapeutic activity and lesser side effects are desired.
ROS such as hydroxyl radicals (HO•), H2O2 peroxyl radicals (R-COO•) and superoxide anion radicals (O2-) are active oxygen components which are short lived by-products of various metabolic reactions. They cause extensive damage to cellular components including DNA, protein, lipids and other biological molecules resulting in many pathological processes such as aging, cancer, cardiovascular diseases, diabetes, inflammation and neurodegenerative diseases. However, biological systems can protect themselves against harmful effects of ROS and free radicals by the formation of antioxidants (Asghar and Masood, 2008).
Antioxidant defense system includes enzymes such as SOD, CAT, and GPx and also contains non-enzymatic agents with endogenous and exogenous sources such as uric acid, bilirubin, vitamin C, carotenoids, polyphenols, etc. Majority of the diseases in humans are believed to be caused due to the imbalance between oxidative stress and anti oxidative defense, thus it is possible to restrict oxidative tissue damage and hence avert disease progression by suitable antioxidant defense supplements (Liu et al, 2003).
The aim of the current project work is to look for specific ACE inhibitors and antioxidants of phytochemical origin from the extracts of the leaves and seeds of Raphanus sativus L. and seeds of Apium graveolens L. Literatures suggest that leaves of Raphanus sativus L. possess gut stimulatory (Ghayur and Gilani, 2005) and hepatoprotective (Ahmad and Anwar, 2006) activity. Seeds of Raphanus sativus L. possess anti-microbial (Rani et al., 2008) and diuretic activity (Mirza, 2003) and seeds of Apium graveolens L. possess hepatoprotective (Anuba and Handa, 1995) and anti-microbial (Misic et al., 2008) activity.
All extracts inhibited ACE in a concentration-dependent manner. The in vitro inhibition of ACE by the seed extract of R. sativus and A. graveolens were moderate when compared to the standard drug, lisnopril. However, at higher doses of the extracts, ACE would be significantly inhibited. Flavonoids are a group of polyphenolics compounds, which have been reported to possess ACE inhibitory activity. Thus, the presence of phenolic and flavonoid content in the extract would have contributed towards ACE inhibition.
The hydroxyl radical is an exceedingly reactive free radical which has been implicated as an extremely damaging species of ROS in free radical pathology, capable of damaging almost all molecules present in living cells. It can join nucleotides in DNA as well as cause strand breakage which ultimately lead to carcinogenesis, mutagenesis and cytotoxicity. The hydroxyl radical scavenging capacity of an extract gives a direct relation to its antioxidant activity. The ability of the extracts to inhibit hydroxyl radical mediated deoxyribose damage was determined by means of the Fe2+-dependent deoxyribose assay. Malondialdehyde, a degradation product of deoxyribose produces a pink chromogen with thiobarbituric acid (Chaudhury et al, 2010). The plant extracts scavenged the hydroxyl radicals present in the reaction mixture and the degradation of deoxyribose was prevented.
NO is a potent pleiotropic agent involved in various physiological processes like smooth muscle relaxation, inhibition of platelet aggregation and regulation of cell mediated toxicity. However, over production of NO manifest in various pathological conditions basically by formation of peroxynitrites. In the nitric oxide scavenging assay, sodium nitroprusside upon interaction with the oxygen present in the saline buffer solution produce nitrite ions that is estimated by Griess reagent (Nagulendran et al, 2007). The plant extracts evaluated were found to decrease the quantity of nitrite ions in vitro which can be attributed to the antioxidant constituents present in the extracts.
The reducing power ability of the plant extracts were found out by measuring the transformation of Fe3+ into Fe2+. The reducing power ability of a compound usually depends on the existence of reductones, which mainly act by breaking the free radical chain reaction by donating a proton (Gupta et al, 2004). The antioxidant principles present in the plant extracts caused the reduction of ferric complex into its ferrous form, and thus accounting for the reducing power ability.
Superoxide anions are spontaneously formed by acquiring an electron from other molecules and in turn damage other bio-molecules either directly or indirectly by forming H2O2, OH-, peroxynitrite or singlet oxygen during aging and pathological conditions like ischemic reperfusion injury. The superoxide anion radical scavenging activity of the extracts was assayed by the PMS-NADH system where the system produces superoxide radicals which reduce the NBT to form a chromophore (diformazan) that absorbs at 560 nm. Determination of the resulting absorbance provides a measure of the level to which the test sample is able to inhibit NBT reduction by the superoxide radical. The plant extracts that were evaluated exhibited good superoxide anion scavenging activity in a concentration dependent manner (Hazra et al, 2008).
Ferrozine on reaction with ferrous ions develops a red coloured complex and in the existence of a chelating agent the complex formation is interrupted and as a result the complex formation is decreased. Thus metal chelating ability of the extracts was denoted by the extent of ferrous-ion-ferrozine complex formation (Hazra et al., 2008). It was reported that the chelating agents that can form σ bond with a metal, are most efficient as secondary antioxidants because they decrease the redox potential and thus steady the oxidized form of the metal ion. The current study shows that the extracts have good ability for Iron binding and thus signifying its antioxidant activity.
Phenolics are secondary metabolite in plant kingdom found in great abundance. These diverse groups of compounds have received much attention as potential natural antioxidant in terms of their ability to act as both efficient radical scavengers and metal chelator. It has been reported that the antioxidant activity of phenol is principally due to their redox potentials, hydrogen donors and singlet oxygen quenchers. Total phenolic assay using folin-ciocalteu reagent is an easy, suitable and reproducible method. It is employed regularly in studying phenolic antioxidants (Nagulendran et al., 2007).
Flavonoids are water soluble pigments present in the cytosol or stored in the vacuole of the plant cell and represent the largest group of phenolic compounds in plants. They exhibit numerous biological effects like antihepatotoxic, anti-ulcer, antiviral, anti-inflammatory, anticancer, anti-allergic activities. They interact with the cardiovascular system in several ways like, reducing oxygen species, increasing nitric oxide concentration and also by inhibiting ACE mainly by binding to the Zn2+ at the active site. Flavonoids due to the presence of their phenolic hydroxyl groups are highly capable of scavenging ROS and are known to be potent antioxidants (Ingrid, 2009). The presence of phenolic and flavonoid content in the extracts has played a vital role in the antioxidant activity by neutralizing the free radicals.
On the basis of the results of this current study, it is clear that the seed extract of R. sativus L., A. graveolens L. and leaf extract of R. sativus L. possess ACE inhibitory and antioxidant activities. ACE inhibitory activity might be helpful in controlling hypertension in patients and thus prevent the progress of several cardiovascular disorders. Free radical oxidative stress have known to play a credible role in the pathogenesis of number of diseases has shown the way to the use of agents that can complement the natural antioxidant defenses. Based on the activity of the extracts through various in vitro assays, it can be concluded that these extracts have significant antioxidant activities.
The various antioxidant mechanisms of the extracts can be related to their strong metal chelating ability, reducing power ability and their effectiveness as scavengers of hydroxyl, superoxide and nitric oxide. The above results show that the seed extract R. sativus have a stronger antioxidant activity than the other two extracts.
In conclusion, the study suggest that the seed extract of Raphanus sativus and Apium graveolens possess angiotensin converting enzyme inhibitory and antioxidant activities that might be helpful in treating hypertension and preventing or slowing the progress of cardiovascular disorders and free radical related disorders. Further investigations on the isolation of active compounds present in the extracts and in vivo studies are necessary to identify a potential chemical entity for clinical use in the treatment of hypertension and other related cardiovascular disorders.
