In Vitro Antioxidant and Inhibitory Effects of Myristica fragrans, Illicuim verum, Curculigo orchioeides, Glycyrrhiza glabra and Embelia ribes against Lipid Peroxidation in Mice Liver.
Summary: The study was designed to assess and compare the antioxidant properties of plants namely, Glycyrrhiza glabra, Illicuim verum, Myristica fragrans, Curculigo orchioeides, and Embelia ribes. To demonstrate lipid peroxidation in mice liver, extract - sodium nitroprusside (5uM) or ferrous sulphate (10uM) was introduced to produce thiobarbituric acid reactive species (TBARS). All plant extracts showed lipid peroxidation inhibitions as revealed by decrease in TBARS. The higher decrease in lipid peroxidation i.e greater antioxidant activity was observed in G. glabra while E. ribes showed relatively weak antioxidant activity. The order of their decreasing lipid peroxidation inhibitory effect and antioxidant activity was G. glabra > I. verum > M. fragrans > C. orchioeides > E. ribes.
Their scavenging activity on DPPH, reducing power and total phenolic contents were also determined which had shown high antioxidant activity and the order of their antioxidant activity was found same as for lipid peroxidation inhibitory effect. The higher inhibitory effect of G. glabra may be due to high phenolic compounds capable of having reducing ability and free radical scavenging activities. Thus, the antioxidants in medicinal plants could be used to oppose oxidative stress in liver and reduce the risk of many free radical related diseases.
Key Words: Antioxidant activity, Medicinal plants, Lipid peroxidation, Oxidative stress, mice liver.
Since ancient times, different parts of the plants were used as medicines for ailments and various diseases . They contain natural products with particular physiological effect . Several kinds of phenolics presents in the plants act as antioxidants [3, 4]. Phenolics can be divided into two groups, simple phenols and polyphenols . The simple phenols are phenolic acids (phenols with carboxyl group) which include hydroxycinamic acids e.g. chlorogenic acid and ferulic acid, etc. and hydroxybenzoic acids, for example, vanilic acid and gallic acid, while polyphenols contain at least two phenol rings which are joined together with a short three carbon chain. Polyphenols are flavonoids  which consist of catechin, rutin and quercetin. These are very important and they are widely used in food industry .
Flavonoids are categorized chalcone, isoflavones, flavone, flavonol, flavanone and anthocyanins. The most important phenolic compounds are described as gallic acid, catechin and rutin sodium carbonate etc. . Their antioxidant activity is due to their redox properties functioning through single oxygen quenching, free radical scavenging and metal ion chelating etc., . During the metabolism, free radicals are produced which cause oxidations resulting in a cell damage and lead to propagation of many diseases [10, 11]. The damaging effects of free radicals are opposed by antioxidants which act as inhibitors of the oxidation processes and protect the human bodies from various diseases, for examples, mutation and cancer etc. .
In our earlier report we have described the bioactive constituents in G. glabra, I. verum, M. fragrans, C. orchioeides, and E. ribes, their pharmacological effects and medicinal uses . These plants have various antioxidants which possess free radical scavenging activity and have protection ability against lipid peroxidation. In present study, antioxidant activity against lipid peroxidation in mice liver and DPPH scavenging activity was undertaken so the antioxidants present in medicinal plants could be used to oppose oxidative stress in liver and could be used to treat liver toxicities.
Thiobarbituric acid (TBA), Butyl hydroxytoluene (BHT), 2, 2-diphenyl-1-picrylhydrazyl (DPPH), ferrous sulphate, sodium nitroprusside (SNP), sodium dodecyl sulphate (SDS) and malonaldehyde-bis-dimethyl acetal (MDA) were obtained from Merck, Sigma-Aldrich and Fluka. All other chemicals used were of analytical grade.
Preparation of Plant Extract
Roots of mulethi (G. glabra), seeds of badian-i-khatai (I. verum), seeds of jaiphal (M. fragrans), roots of musli siah (C. orchioeides) and fruits of baberang (E. ribes) were purchased from Pansari shops and authenticated by a botanist, Faculty of Medical and Health Sciences, University of Poonch, Rawalakot Azad Kashmir, Pakistan. The parts of plants used in our experiments were dried and ground to fine powders and then suspended in water. The extracts were prepared as mentioned in our earlier report . Dilutions were made to obtain various concentrations (25-400 ug/mL) of each extract for the experiment. Albino mice were supplied by National Institute of Health, Islamabad and handled strictly in accordance with NIH guidelines.
Production TBARS on Lipid Peroxidation and its Estimation
In vitro, production of TBARS as a marker of lipid peroxidation was done by a method as described by Ohkawa et al . The mice were anaesthesized with ether and the liver was immediately collected in ice box. One gram liver was cut into small pieces and homogenized in a Waring blender for 2 min with 1 volume of water. The homogenate was centrifuged at 3840x g for 10 min. The supernatant collected was used for the determination of TBARS produced by prooxidants which was as follows: To 0.1mL of supernatant, 0.05mL of 15uM nitroprusside or 0.05mL of 30uM ferrous sulfate as pro-oxidant was added, followed by the addition of 0.015mL of plant extract to obtain final concentrations of 50 to 250ug/mL. The final volume was adjusted to 0.3 mL with water and incubated for one hour at 37o C.
Similarly control was prepared in which pro-oxidant was omitted while in blank (Basal), both pro-oxidant and plant extract were omitted. In each case, the color reaction was performed by adding 0.2mL, 0.2mL and 0.5mL of each of the 8.1% sodium dodecyl sulphate (SDS), acetate buffer, pH 3.4 and 0.6% TBA, respectively. The reaction mixtures were incubated at 97o C for 1 hour. After cooling it was then diluted appropriately with 0.03 mM MDA and the absorbance of each tube was recorded at 532 nm.
Antioxidant activity by DPPH Radical Scavenging
Antioxidant activity of different extracts was determined according to the Kulisic et al [15 ]. To 950uL of 86.5 umol methanolic solution of DPPH, 50uL of plant extract was added to obtain final concentrations ranging from 25 to 750ug/mL. The solution was vortexed and kept for one hour at room temperature. A control was prepared in which extract was replaced by methanol. The absorbance was measured specrophotometrically at 517 nm.
The DPPH scavenging activity was expressed as inhibition percentage as follows.
DPPH % inhibition = [(AB - AS)/ AB x 100
Where AB is the absorbance of the control and AS is the absorbance of the extract sample. BHT was used as positive control as shown in Fig. 1 and concentration of each extract produces half of maximum %DPPH scavenging activity (IC50) was calculated from graph.
Total Antioxidant Activity
Total antioxidant assay is based on the reduction of phosphomolybdic acid (MoVI) to green coloured complex of phosphomolybdenum (MoV ) which was performed by the method of Prieto et al . 5-20ug of extract in 50-200uL of water was added to 3mL of the reagent solution ( prepared by dissolving 10.6g of sodium phosphate, 60mL of sulfuric acid and 4.9g of ammonium molybdate in one liter of water). The mixture was incubated for 1.5 h at 95 0C. After cooling, absorbance was noted at 695 nm against reagent blank. Standard calibration curve (0-25 ug of ascorbic acid /mL) was constructed following above procedure and antioxidant capacities were expressed as equivalents of ascorbic acid/mL.
Determination of Total Phenolic Content
The total phenolic content of various extract samples was determined by using Folin reagent method . The detail procedure for the determination of total phenolic content is described by Bhatti et al . Standard calibration (0-25ug of gallic acid /mL) curve was constructed following the same procedure. The results were expressed as mg of gallic acid equivqlents (GAE)/g dry weight extract.
Results and Discussion
Liver homogenates were treated with 5uM SNP or 10uM iron to create lipid peroxidation, resulting in a production of certain level of TBARS and the effects of extracts from G. glabra, I. verum, M. fragrans, C. orchioeides and E. ribes on lipid peroxidation were determined.
Fig. 1 shows the effects of these plant extracts on mice liver homogenate induced with 5uM sodium nitroprusside on lipid peroxidation. The treatments with each plant extract of various concentrations (50-250 ug/mL) decreased lipid peroxidation as revealed by decrease in TBARS level. G. glabra and I. verum exhibited greater lipid peroxidation inhibitions accompanied by higher decrease in TBARS production. Thus, both plants have higher antioxidant activities. Other three plants, M. fragrans, C. orchioeides and E. ribes showed relatively weaker antioxidant activities because gradual decrease in lipid peroxidation inhibitions and gradual increase of TBARS productions were found, respectively. Fig.1 indicates that in all five plants, lipid peroxidation inhibitory effect and antioxidant activity were increased as the concentrations of extract increased.
Thus, the order of their decreasing lipid peroxidation inhibitory effect and antioxidant activity was G. glabra > I. verum > M. fragrans > C. orchioeides > E. ribes.
Fig. 2 also shows the antioxidant effect of these plants in homogenate induced with 10uM FeSO4. Iron produced certain amount of TBARS compared to the basal (130 nmol TBARS/g tissue). However, plant extracts caused a decrease in lipid peroxidation due to their antioxidant property of the plant extracts. In all plant extracts, the TBARS productions were decreased progressively as the concentrations of the extract increased from 50 to 250ug/mL. In other words all five plants showed a trend of regular decrease in lipid peroxidation as the concentrations of the extracts increased. G. glabra exhibited greater lipid peroxidation inhibition and higher antioxidant activity while E. ribes showed smaller lipid peroxidation inhibition and less antioxidant activity.
Thus, the order of their decreasing lipid peroxidation inhibitory effect and antioxidant activity was G. glabra > I. verum > M. fragrans > C. orchioeides > E. ribes Fig. 3 shows the DPPH scavenging activity of plants. All plants have shown high antioxidant activity. G. glabra and I. verum have shown greater DPPH % scavenging activity than that of M. fragrans, C. orchioeides and E. ribes which is also evident by their IC50 values (Table-1). The same order of their decreasing antioxidant activity was observed.
Table-1: IC50 values of various extracts of medicinal plants assessed by DPPH.
Table-2 shows the total antioxidant activity of different plants extracts expressed as ascorbic acid equivalents. Antioxidant activity was termed as reducing capacity and all the extracts showed their reducing power. As the concentrations of the extracts increased, the reducing capacities were also increased. However, the order of their decreasing reducing activity was similar to the pattern of lipid peroxidation inhibitory effect.
Table-2: Total antioxidant activity of different plants extracts measured by phosphomolybdenum reduction method
Plant/ Concentrations###5ug extract###10ug extract###20ug extract
Total phenolic contents are shown in Table-3. The results revealed that the highest phenolic content was observed in G. glabra (339 mg GAE/g) and I. verum (319. mg GAE/g) followed by M. fragrans (293 mg GAE/g), C. orchioeides (287 mg GAE/g) and E. ribes (263 mg GAE/g /g). The order of their decreasing total phenolics in plants was also found same as mentioned above in all cases.
Table-3: Total phenolic content among aqueous extract of medicinal plants
###Extract###Phenolics (mg GAE/g of extract)
Antioxidant activities in the plant extracts is due to the presence of some phenolic acds and flavonoids which mediates as reducing agents, to scavenge reactive oxygen species and chelate trace metals etc., . G. glabra, I. verum, M. fragrans, C. orchioeides and E. ribes were chosen as they are frequently used as antihypercholestermic agents in a number of herbal formulations. Sodium nitroprusside is a drug for reducing blood pressure which dilates the arterioles and veiniols. Its cytotoxicity is due to the production of nitric oxide . In mice liver, methionine synthase is deactivated by nitric-oxide which damages the cell structures (proteins, lipids, membranes, DNA) and is simply called oxidative stress. Sodium nitroprusside or iron as pro-oxidants stimulate the production of TBARS in mice liver and cause lipid peroxidation.
Aqueous extracts of these five plants have shown inhibition against lipid peroxidation by decreasing the production of TBARS in mice liver. So these plants may be used to prevent the diseases arising from sodium nitroprusside or iron overload. The order of their decreasing lipid peroxidation inhibitory effect was G. glabra > I. verum > M. fragrans > C. orchioeides > E. ribes. The same results were obtained with mice brain. But the order of their decreasing lipid peroxidation inhibitory effect and antioxidant activity was little different . G. glabra and I. verum exhibited greatest antioxidant activity against peroxidation created by sodium nitroprusside and iron. These results were also proved by total phenolic contents because the G. glabra contained 339 mg GAE/g of extract and I. verum have 319 mg GAE/g of extract, the highest phenolic contents among all five plants.
DPPH scavenging activity at IC50 values of G. glabra (26ug/mL extract) and I. verum (42 ug/mL extract) and total antioxidant assay of 88 and 72 ug ascorbic acid equivanlent /mL of extracts of both plants also proved that G. glabra and I. verum have highest anti-oxidant activity. The other three plants, M. fragrans, C. orchioeides, and E. ribes showed relatively weak antioxidant activity due to relatively low phenolic contents, less scavenging activity and small total antioxidant activity (Tables 1-3). Many biologically active compounds such as flavonoids, triterpene, saponins and pectin etc, from G. glabra root were reported to have antioxidant effects [20, 21]. Murcia et al  and Olaleye et al  have demonstrated M. fragrans have antioxidant property due to phenolic compounds. It has been reported to show DPPH free radicals, cation radicals ABTS and hydroxyl (HO-1.) radicals scavenging activity and ability to chelate with metal ions [24, 25].
Chatterjee et al  also reported phenolic compounds in M. fragrans which have great potential to inhibit lipid peroxidation. Aqueous extract of I. verum possessed antioxidant activity against H2O2 induced DNA damage. The antioxidant activities were evaluated by various methods such as TBARS assay, DPPH assay ,phosphomolybdenum assay, hydroxyl radical scavenging assay and superoxide anion radical scavenging assay . Devyani et al  examined the antioxidant activity of C. orchioides and revealed that the extract had DPPH, nitric oxide and superoxide scavenging activity and had protection against lipid peroxidation. E. ribes have been reported to exhibit DPPH free radical scavenging activities .
However, protection against free radicals by the extracts indicates that these can be used to treat liver and brain toxicities.
The inhibitory effect and antioxidant activity of medicinal plants, which studied in this research may be due to presence of higher phenolic contents, free radical scavenging activity and reducing ability. These plants are perfect sources of antioxidants and may be used to prevent oxidative stress in liver.
This research work was done to complete M.Phil thesis in the Department of Biological Sciences, Gomal University, D.I.Khan, Pakistan.
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|Author:||Saeed, Asma; Rehman, Saif Ur; Raza, Ali; Abbas, Ali; Naz, Rubina; Jan, Saleem; Saeed, Ahmad|
|Publication:||Journal of the Chemical Society of Pakistan|
|Date:||Oct 31, 2017|
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