Volume 6, Issue 2, April 2016, Pages 153–159
Open Access
Original article
- Open Access funded by Center for Food and Biomolecules, National Taiwan University
- Under a Creative Commons license
Abstract
Reactive
oxygen and nitrogen species (RONS) are involved in
deleterious/beneficial biological processes. The present study sought to
investigate the capacity of single and combinatorial herbal
formulations of Acanthus montanus, Emilia coccinea, Hibiscus rosasinensis, and Asystasia gangetica
to act as superoxide radicals (SOR), hydrogen peroxide (HP), nitric
oxide radical (NOR), hydroxyl radical (HR), and
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical antagonists using in vitro
models. The herbal extracts were single herbal formulations (SHfs),
double herbal formulations (DHfs), triple herbal formulations (THfs),
and a quadruple herbal formulation (QHf). The phytochemical composition
and radical scavenging capacity index (SCI) of the herbal formulations
were measured using standard methods. The flavonoids were the most
abundant phytochemicals present in the herbal extracts. The SCI50
defined the concentration (μg/mL) of herbal formulation required to
scavenge 50% of the investigated radicals. The SHfs, DHfs, THfs, and QHf
SCI50 against the radicals followed the order
HR > SOR > DPPH radical > HP > NOR. Although the various
herbal formulations exhibited ambivalent antioxidant activities in terms
of their radical scavenging capabilities, a broad survey of the results
of the present study showed that combinatorial herbal formulations
(DHfs, THfs, and QHf) appeared to exhibit lower radical scavenging
capacities than those of the SHfs in vitro.
Graphical abstract
Radical scavenging capacity indices (SCI50) of single and combinatorial herbal formulations.
Keywords
- antioxidant;
- herbal formulation;
- in vitro;
- phytochemicals;
- radicals
1. Introduction
Reactive
oxygen and nitrogen species (RONS) or radicals and oxygen derived,
nonradical reactive species (nRRS), referred to as pro-oxidants, are
involved in deleterious/beneficial biological processes such as
mutation, aging, carcinogenesis, degenerative diseases, inflammation,
signal transduction, immune response, cellular regulatory events, and
cell development.1, 2, 3, 4, 5, 6, 7, 8 and 9 Both RONS and nRRS are predictable products of aerobic metabolic pathways10
that encompass membrane-bound reduced nicotinamide adenine dinucleotide
phosphate (NADPH)-dependent oxidase, lipoxygenase, cytochrome P-450,
and xanthine oxidase activities.9 and 11
Numerous reports have shown that oxidative stress injuries are metabolic outcomes of noxious chemical agents12 and 13 or impaired metabolic events,14 and 15
which are characterized by disequilibrium between physiologic levels of
oxidants and corresponding activities of antioxidant systems. The RONS
include among other reactive oxides, the superoxide ion (O2−), nitric oxide (NO−), hydroxyl (OH−), peroxyl (ROO−), and alkoxyl (RO−), whereas the nRRS and their derivatives include hydrogen peroxide (H2O2), organic peroxide (ROOH), hypochlorous acid (HClO), Ozone (O3), aldehydes (RCOH), peroxynitrite (ONOOH), and singlet oxygen (1O2).5 and 9
Depending on its prevailing environmental pH, superoxide may exist in two states as O2− (high pH) or hydroperoxyl (HO2·) (low pH) ion, which defines its biologic properties.5 and 16 Evidence showed that at acidic pH the most important reaction of O2− is dismutation.5 The O2− is a powerful nucleophile, capable of attacking positively charged centers of array of biomolecules. As an oxidizing agent, O2−
reacts with proton donors such as ascorbic acid and tocopherol.
Conversely, when present in organic solvents, its ability to act as a
reducing agent is increased.5
Spontaneous dismutation of O2− or/and superoxide dismutase (SOD) activity is the primary generator(s) of cellular H2O2.5 and 17 The deleterious actions of H2O2
stems from its oxidizing potential and its ability to act as a
substrate for the generation of other oxidizing species, such as OH− and HClO.18 and 19 The molecular bases of H2O2 toxicity include their capability to degrade heme proteins, inactivate enzymes, oxidation of DNA, lipids, and SH groups.17 and 20
The NO− is produced by the oxidation of one of the terminal guanido nitrogen atoms of l-arginine. The nitric oxide synthase (NOS) pathway is responsible for the biosynthesis of NO− in a variety of tissues.19
The presence of endotoxins and/or cytokines in mononuclear phagocytes
induces NOS, the so-called iNOS, which elicits raised cellular levels of
NO−.21 The NO−
derivative-ONOOH, elicits the depletion of SH groups and oxidation of
biomolecules, engendering tissue damage similar to that caused by the
actions of OH−, such as DNA damage, protein oxidation, and nitration of aromatic amino acid residues in proteins.22
The formation of OH− accounts for much of the damage done to biological systems by increased generation of O2− and H2O2.23 The most important biological properties of OH− are abstraction, addition, and electron transfer reactions.19 Generally, OH− is a fast reacting and powerful oxidizing agent. According to in vitro studies by Cohen, 12 certain cell toxins effect their deleterious actions on specific target cells through intracellular generation of OH−. In physiologic systems, reactions of OH−
with biomolecules such as DNA, proteins, lipids, amino acids, sugars,
and metals are the biochemical bases of several pathologic disorders and
the ageing process. 6 and 24
The
2,2-diphenyl-1-picrylhydrazyl (DPPH) is a stable free radical used for
ascertaining the capacity of tissue extracts to act as free radical
scavengers and to measure their antioxidant activity in vitro. 25, 26 and 27 The reaction of DPPH with antioxidant of tissue extracts produces a corresponding reduced compound (hydrazine DPPH2), which can be monitored by color change from purple to yellow with maximum absorptivity (ƛmax) within the range of 515–528 nm 28 and 29
The medicinal usefulness of Acanthus montanus, Emilia coccinea, Hibiscus rosasinensis, and Asystasia gangetica has been reported elsewhere. 30, 31, 32, 33 and 34
Most of the therapeutic benefits derivable from medicinal plants are
hinged on their capability to ameliorate oxidative stress. 35, 36, 37 and 38
Furthermore, alleviation of oxidative stress-induced pathologic
conditions following the administration of RONS antagonists from diverse
plant species have been reported by several authors. 39, 40 and 41
Accordingly, most ethnomedicinal practices presume that poly-herbal
decoctions are more efficacious than mono-herbal formulae against
pathologic conditions and physiologic disorders. 26, 42, 43, 44 and 45
However, combinatorial herbal formulations have been reported to cause
alterations in the pharmacologic properties and therapeutic outcomes of
individual plant extracts. 26, 29 and 45 The present study sought to investigate the capacity of single and combinatorial herbal formulations of A. montanus, E. coccinea, H. rosasinensis, and A. gangetica to act as RONS and nRRS antagonists using in vitro models.
2. Materials and methods
2.1. Collection and preparation of herbal samples
Fresh leaves of A. montanus (Nees) T. Anderson (ACMO), E. coccinea (SIMS) G. Don (EMCO), and H. rosasinensis
L. (HIRO) were collected from uncultivated lands in Umuamacha Ayaba
Umaeze, Osisioma Ngwa LGA (Local Government Area), Abia State, Nigeria,
whereas fresh leaves of A. gangetica L.T. Anderson (ASGA) were
collected from Ubowuala, Emekuku, Owerri North LGA, Imo State, Nigeria.
The four herbs were identified and authenticated by Dr. M. Ibe, School
of Agriculture and Agricultural Technology (SAAT), Federal University of
Technology, Owerri. All the leaves were collected between the months of
July and August, 2009.
The
leaves of individual plants were washed with continues flow of
distilled water for 15 minutes and allowed to dry at laboratory ambient
temperature (24 ± 5 °C). A 500-g part of each herbal sample was weighed
using a triple beam balance (OHAU 750-50; OHAUS Triple Beam Balance,
Model TJ611, Burlington, NC, USA) and dried in an oven (WTC BINDER; 7200
Tuttlingen, Germany) at 60 °C until a constant weight was achieved. The
dried leaves were packaged in dark polyethylene bags and kept in a cold
room (7 ± 3 °C) for 24 hours before pulverization. Next, the separate
dried leaves were pulverized using the Thomas-Willey milling machine
(ASTM D-3182; India), after which the ground samples were stored in
air-tight plastic bottles with screw caps pending extraction.
2.2. Extraction of herbal samples
A
portion of 40 g of each pulverized dried sample of ACMO, ASGA, EMCO,
and HIRO were subjected to repeated soxhlet extraction cycles for 2
hours using 96% C2H5OH (BDH, UK) as solvent to
obtain a final volume of 500 mL of each herbal extract. The volumes of
the extracts were concentrated and recovered in a rotary evaporator
(Rotavapor R-200; Büch, BÜCHI Labortechnik AG, Flawil, Switzerland) for
12 hours at 60 °C under reduced pressure. The extracts were dried in a
desiccator for 24 hours, wrapped in aluminum foil, and stored in
air-tight plastic bottles with screw caps at ≤ 4°C. The yields were
calculated to be as follows: ACMO = 16.35% (w/w), ASGA = 16.69% (w/w),
EMCO = 17.99% (w/w), and HIRO = 17.23% (w/w). The separate herbal
extracts were reconstituted in phosphate-buffered saline (PBS) solution,
osmotically equivalent to 100 g/L PBS (90.0 g NaCI, 17.0 g Na2HPO4·2H2O and 2.43 g NaH2PO4·2H2O). Portions of the individual extracts were also measured for phytochemical contents.
