Evaluation of in vitro antioxidant, reducing, lipoxygenase and ACE inhibition activity of polyherbal drug linkus

Mahreen Latif
Multidisciplinary Research Laboratory (MDRL), Bahria University Medical and Dental College, Karachi-75500, Pakistan.
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Khan Usmanghani

Khan Usmanghani
Herbion Pakistan (Pvt.) Ltd., Korangi Industrial Area, Karachi-74900, Pakistan.
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Article Number - D88AB1061983
Vol.10(44), pp. 945-953 , November 2016
https://doi.org/10.5897/AJPP2015.4520

Received: 29 December 2015
Accepted: 18 February 2016
Published: 29 November 2016

Copyright © 2024 Author(s) retain the copyright of this article.
This article is published under the terms of the Creative Commons Attribution License 4.0.

ABSTRACT

Oxidative, reductive, lipoxygenase and angiotensin converting enzyme (ACE) activities are the condition where there is an inequity among concentrations can cause a multiple pathological effects. Vast results of medical plants and the remarkable contribution on humans has been observed since centuries. The current study has showed the antioxidant, lipoxygenase, ACE inhibition, urease activity and reducing ability in vitro on linkus formulation, including lozenges and syrup. The anti-oxidant activities was assessed by scavenging ability of the linkus on free radical (DPPH; C₁₈H₁₂N₅O₆) 2,2’-diphenyl-1-picryl hydrazyl. For lipoxygenase measurements, purified lipoxygenase with lionoleic acid as substrate was used for the activity, however for lipoxygenase activity the thiocholine and diothiobisnitrobenzoic acid (DTNB) was used. Indophenol method has been used for determination of urease activity, however the reducing ability has been assessed by the conversion of ferric into ferrous state. In vitro results of linkus syrup as compared to standard showed good anti-oxidant and reducing ability. Moderate activity of urease, lipoxygenase and ACE inhibition were observed with comparison of standard. These activities of polyherbal formulation might be helpful for reducing cough and related symptoms.

Key words: Antioxidant activity, reducing ability, urease activity, lipoxygenase and ACE inhibition, linkus.

INTRODUCTION

Reactive oxygen species (ROS) is the byproduct of oxidative stress under the physiological conditions. These extreme ROS accretion will lead to cell damage, such as damage to proteins, DNA, and lipid membranes. The cell injury/damage is initiated by ROS and has been associated with the development of numerous disease injury/damage is initiated by ROS and has been associated with the development of numerous disease conditions, such as diabetes, atherosclerosis, cancer and cardiovascular disease etc. Its comprising multiple free radicals and exogenous factors play vital role (Finkel et al., 2000).

ROS in vivo inside the cell membrane performs multiple mechanism by sunlight or by different chemical and metabolic process, including DNA damage, carcinogenesis etc (Gyamfi et al., 1999; Ganapathy et al., 2011; Gutteridge and Halliwell, 2000; Halliwell, 2001). The free radical, chemicals and toxins creates effects in immune system and are declared as the major contributor of free radicals in the oxidation process (Halliwell, 1994; Kuhnan, 1976; Kumpulainen and Salonen, 1999; Younes, 1981).

Urease is the prominent agent for gastrointestinal track (GI) and help to inhibit Helicobacter pylori. Urease also acts directly as virulence factor in infections other than GI, including urinary tract both in humans and animals (Ghous et al., 2010; Halliwell et al., 2008; Nabati et al., 2012). Lipoxygenase is the significant main enzyme for biosynthesis of leukotriene and different potential drug to cope with asthma, arthritis, circulatory diseases etc (Wasserman et al., 1991) (incorporated inside text). Consequently, over a decade, the foremost effort has invested and many vitro active like 5-lipoxygenase inhibitors has developed (Ford-Hutchinson, 1991; Batt, 1992; McMillan and Walker, 1992; Ford-Hutchinson et al., 1994). ACE inhibitors are known to induce dry cough. There has an observation that prostaglandins with others are responsible for this effects but the statement is still controversial (Morice et al., 1987; Gilchrist et al., 1989; Fox et al., 1996).

Naturally occurring antioxidants have effective pharmacological action, including less toxicity and price effectiveness. Multiple plant products, including terpines and phenols also have this activity too (De Souza et al., 2007; Lin and Yin, 2007; Rice-Evans et al., 1996). This present study was based on linkus syrup and lozenges which have a wonder blend of polyherbs, including Glycyrrhiza glabra, Adhatoda vasica, Viola odorata, Piper longum, Hyssopus officinalis and Alpinia galangal (Appendix 1 and 2). The study focused on anti-oxidant and reducing ability on linkus dosage forms and proved the strength of activity inside the herbal formulation (Figure 1). By proving the activities, it claims that linkus might work on associated symptoms of cough and respiratory tract.

METHODOLOGY

Plant material and techniques

Herb extracts and mentioned chemical constituents were the part of Linkus lozenges and Syrup (Figure 2). Major specification included organoleptic evaluation, qualitative reaction of glycyrrhizic acid, tanning agents and ascorbic acid. Quantitative determination with spectrophotometric evaluation was observed. The total flavonoids contents as luteolin-7-glucoside were not less than 0.080 mg/lozenge (Zeeshan et al., 2014).

Preparation of plant extract

Individual herbs were taken separately, cleaned, grind, weighed and distill water added together with sugar and liquid glucose. Ingredients were transferred from weighing tank to storing tank with temperature range from 110 to 120°C for syrup and 60 to 80°C for lozenges. For lozenges, boiling and vacuum cooking was needed. Kneading, roping and sizing were the next step with the help of uni-plast machine. Lozenges were finally passed through cooling tunnel to obtain the desired hardness. For syrup, hot water filtrate was evaporated via a condenser. Methyl paraben, and Propyl paraben together with flavoring agents were added after completion (Zeeshan et al., 2014)

Chemicals and Reagents

All chemicals were high performance liquid chromatography (HPLC) grade. For reduction, 1, 1-diphenyl-2-picrylhydrazyl was used and obtained from Merck, Pakistan, 2,2’-diphenyl-1-picryl hydrazyl (DPPH) was obtained from Sigma-Aldrich Chemie (Buchs, Switzerland) and used for anti-oxidant activity.

Scavenging activity by DPPH radical

The antioxidant activity was measured by the scavenging aptitude of the syrup and capsules on free radical (DPPH). Antiradical activity analyzed depended on the reduction of DPPH. Its free radicals showed strong absorption at 517 nm due to odd electrons. When this electron was paired in the company of hydrogen donor, for example any antioxidant, the absorption strength decreased and color changed from purple to yellow, with respect to the number of electrons captured (Gülçin et al., 2005). For performing the process, 2, 2-Diphenyl-1-(2, 4, 6-trinitrophenyl) hydrazyl (M.W = 394.24) (Sigma) was prepared in ethanol in a concentration of 3 mM. Each well in 96-well plate was labelled as control, blank and test compound of various concentrations. DPPH solution (95 µl) was added in the labeled wells. The test compound (5 µl) of concentration 10 to 1000 µM in dimethyl sulphoxide (DMSO) was then added in DPPH solution and reaction mixture was mixed for few seconds. The reaction took place in wells when 96 well plates were incubated at 37°C for 30 min. The micro titre plate was read at the absorbance of 515 nm (Spectramax plus 384 Molecular Device, USA) after 30 min. The percentage of radical scavenging activity was considered with respect to DMSO treated control. Butylated hydroxyanisole (BHA) was taken as standard. The DPPH activity was performed with the help of the following equation:

DPPH radical scavenging effect (%) = Ac – As / Ac × 100”

Where As = absorbance of test compound, Ac = absorbance of control

Reducing ability by the conversion of ferric into ferrous state

The reducing ability was determined by the conversion of ferric into ferrous state by antioxidant compounds using the method of Oyaizu (1986). Each test compound (100 µl: 10 to 1000 µM) prepared in DMSO was mixed with phosphate buffer (250 µl: pH 6.6: 0.2 M). Potassium ferricyanide (250 µl: 1%) was then added to the contents in the test tube. This mixture was then incubated at 50°C for twenty minutes in water bath and was centrifuged for ten minutes at 3000 rpm. Subsequently on centrifugation, the top layer of solute (250 µl) was separated in another set of test tubes and mixed with equal volume of DMSO (250 µl). Ferric chloride (0.1 %: 50 µl) was added to the mixture with absorbance at 700 nm on spectrophotometer (Specord 2000, Germany). Percent reduction ability was determined in terms of percentage with respect to BHA used as standard.

Percent reduction activity = At/As × 100

Where As = absorbance of standard, At = absorbance of test.

Antiulcer/anti urease activity

By using the indophenol method, the urease activity was evaluated by ammonia production as described by Weatherburn. Reaction mixtures encompassing 25 μl of enzymatic (Jack bean Urease) solution and 55 μl of buffers comprising 100 mM urea were incubated with 5 μl of test compounds for 15 min at 30°C in 96-well plates (Tariq et al., 2011). With the help of indophenol method, activity was determined by measuring ammonia production. After that, 45 μl of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 70 μl of alkali reagent (0.5% w/v NaOH and 0.1% active chloride NaOCl) were added with each well. By using a microplate reader (Molecular Device, USA), the absorbance at 630 nm was measured after 50 min. In a final volume of 200 μl, all reactions were performed in triplicate. By using SoftMax Pro software (Molecular Device, USA), the results (change in absorbance per min) were processed. All the assays were performed (0.01 M K₂HPO₄.3H₂O, 1 mM EDTA and 0.01 M LiCl₂) at pH 8.2. Percentage inhibitions were intended from the formula 100 - (OD_testwell/ OD_control) × 100. As the standard inhibitor of urease and Thiourea was used (Khan et al., 2013).

Lipoxygenase inhibition activity

Lipoxygenases are family of iron encompassing dioxygenases that convert the addition of molecular oxygen to fatty acid comprising a cis-1, 4- pentadiene classification. The prime product of this response is a “4-hydroperoxycis trans-1, 3-conjugated pentadienyl moiety” within unsaturated fatty acid. This assay processes the hydroperoxides produced in the lipoxygenation reaction using a purified lipoxygenase with lionoleic acid as substrate (Tappel, 1986: Chedea et al., 2012). In the proposed method, lipoxygenase enzyme solution was prepared in sodium phosphate buffer with such concentration to give 130 U per well. Sodium phosphate buffer (pH 8.0: 160 µl:100 mM) was occupied in each well of plate labelled as Blank named B substrate and B enzyme, as control and Test. Test compound solution in methanol (10 to 1000 mM: 10 ml) was added in each well labelled as test. Lipoxygenase solution (LOX: 20 µl) was added in each well including B enzyme, Control and Test except B substrate and the mixture was incubated at 25°C for ten minutes. Substrate solution was prepared by adding linoleic acid (155 µl:0.5 mM) into 0.12% w/v tween 20 (257 µl). The mixture was mixed and 0.6 ml NaOH (1 N) was added to remove turbidity and volume was made up to 20 ml with deionized water. This mixture was dispersed with the nitrogen gas to evade autoxidation before adding to each other. The response was started by the adding of 10 µl substrate in each well except enzyme B, also the absorbance was measured at 234 nm for 5 min.

RESULTS

Linkus is the poly herbal formulation analyzed for antioxidant, reducing, and lipoxygenase and ACE inhibition activity with different concentration (10, 50, 100 μl/ml) on 2 dosage of different dosage forms, comprising lozenges and syrup. When formulations of syrup and lozenges were compared at various concentrations (10, 50,100 µg/ml), DPPH radical scavenging activity increased in a dose dependent manner for both formulations just like standard BHA as shown in Table 1. It showed that both dosage forms, including syrup and lozenges have good antioxidant potential that is, 23.4, 45.7, 65.0% w.r.t standard BHA. For determining the reducing activity, ferrous were the leading component. Both dosage forms of syrup and lozenges had some reducing ability as compared to standard as shown in Table 2. For protecting the gastric mucosa, the syrup and lozenges have some anti-urease activity too as shown in Figure 3 and Table 3. Lipoxygenase compounds are the derivatives of arachadonic acid. After analysis, it was determined that the lozenges and capsules have some efficacy for the reduction of inflammation. Results are shown in Table 4.

DISCUSSION

Oxidant cause damage to proteins, macromolecules and DNA and this causes many damages in human tissues including aging (Ames et al., 1992; Fraga et al., 1990; Harman, 1981; Sai et al., 1992; Stadtman et al., 1992; Harman, 1992). Currently, available antioxidant compound including butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have negative impact on human health (Barlow, 1990; Branen, 1975). For reducing the impact of oxidants, the natural occurring plants have been used for medical purpose (Schuler, 1990). Various plant species have been explored for antioxidant activity (Chu et al.,, 2000; Koleva et al., 2002; Mantle et al., 2000; Oke and Hamburger, 2002). DPPH is the sensitive method for antioxidant screening for plant extracts (Koleva et al., 2002). For determining the antioxidant activity in poly herbal formulation, scavenging ability was used. Absorption was noted on 517 nm due to odd electron. Good antioxidant activity was observed in both dosage forms. For determination of reducing activity Fe ⁺³ to Fe⁺²was the investigating point (Oyaizu, 1986). For antioxidant activity, the decreasing potential of a compound seems to be a substantial indicator (Meir et al., 1995). Percentage reducing ability was determined by the BHA standard formula and the results found good reducing ability in the poly herbal formulation.

Free radical and lipid per oxide play a vital role for the development of ulcer in human (Gutteridge, 1995). Linkus poly herbal formulation shows a good gastric protection. Herbal formulations have anti-inflammatory activity, including many disorders such as cough, chronic laryngitis and many others (Kapoor, 2000; Madras, 1993). Beside all the functions and activities linkus formulation shows good lipoxygenase inhibition activity (Figure 4).

Cough is the furthermost common today’s symptom seen in overall family practice. Clinically, a cough is nearly a symptom of an underlying illness. It is significant to look beyond it to treat the cause and, hence achieve the maximal relief from cough and related symptoms. Studies have shown that there is an interaction between respiratory tract infections and antioxidant activities (Rubin et al., 2004; Gilliland, 2003) and oxidative stress present in blood due to respiratory infections (Gilliland et al., 2003). Lipoxygenase and leukotriene are the key factors for the inflammatory responses and respiratory distress (Wasserman et al., 1991). Multiple events suggested that lipoxygenase have strong relation in physiological event in respiratory tract infection (Holroyde, 1981; Weiss, 1982; Barne, 1984; Smith, 1985; Adelroth, 1986).

This study has shown the visible antioxidant activity, urease and lipoxygenase activity in poly herbal formulation Linkus cough syrup and lozenges (Figure 4). These dosage forms have contributing factors towards the indication cough as antioxidant and anti-inflammatory activity. These type of activity are due to free radical 2,2’-diphenyl-1-picryl hydrazyl, conversion of ferric into ferrous state, ammonia production using the indophenol method and hydroperoxides produced in the lipoxygenation reaction.

CONCLUSION

The poly herbal extract based lozenges and syrup were analyzed in vitro for anti-oxidant, urease and ACE inhibiting activity. Syrup was found to be more potent in comparison with lozenges but significant ability was found in contrast assessment with standard. It might be helpful for the reduction of respiratory tract infection and allied problems with minimum adverse/side effects.

CONFLICT OF INTEREST

The authors have not declared any conflict of interest.

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