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Antioxidant, antimicrobial and anti-inflammatory activities development of methanol extract of Saxifraga veronicifolia Pers

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  • National institute for Agricultural research, Algeria

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Plant secondary metabolites are excellent candidates for developing new phytopharmaceuticals. Hence, the biological potentials development of Saxifrage veronicifolia extract was evaluated using antioxidant activity (DPPH and reducing power), antimicrobial activity with ATCC strains (Escherichia coli, Proteus mirabilis, Bacillus subtilis , Staphylococcus aureus and Candida albicans ) and anti-inflammatory test with Protein denaturation inhibition power. The results showed good antiradical and inflammatory effects but a low antimicrobial power.
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Antioxidant, antimicrobial and anti-inflammatory activities development of
methanol extract of Saxifraga veronicifolia Pers.
Nouioua Wafa 1 & Gaamoune Sofiane 2
1)Faculty of Natural Life and Sciences, University Ferhat Abbas Setif, Algeria
2)National Institute of Agricultural Research – Setif – Algeria.
Received: April 16, 2019; Accepted: June 3, 2019
Corresponding author Email science1105@hotmail.fr
Copyright © 2019-POSL
DOI:10.163.pcbsj/2019.13.-2-110
Abstract. Plant secondary metabolites are excellent candidates for developing new phytopharmaceuticals.
Hence, the biological potentials development of Saxifrage veronicifolia extract was evaluated using antioxidant
activity (DPPH and reducing power), antimicrobial activity with ATCC strains (Escherichia coli, Proteus
mirabilis, Bacillus subtilis , Staphylococcus aureus and Candida albicans ) and anti-inflammatory test with
Protein denaturation inhibition power. The results showed good antiradical and inflammatory effects but a low
antimicrobial power.
Key Words: Saxifraga veronicifolia; antiradical; antimicrobial; Protein denaturation.
1. INTRODUCTION
Saxifraga Linnaeus is the largest genus within the family of Saxifragaceae [1]. The
Saxifragaceae family includes 540 species, out of which various Saxifraga species account for
more than 80%. Saxifrages are cushion or mat-forming perennials which are distributed in the
subarctic and arctic zones of the Northern Hemisphere, inhabiting primarily higher mountain
belts, as well as in the mountains of North Africa and South America; but some of them also
occur in the tropical zone. Saxifrage species are significantly varied ecologically, though
many of them show adaptations to mountainous, stony or rocky habitats [2, 3].
Most saxifrage is smallish plants whose leaves grow close to the ground, often in a rosette.
The leaves typically have a more or less incised margin; they may be succulent, needle-like
and/or hairy, reducing evaporation.Saxifraga veronicifolia havelobed crenate basal leaves. 10-
30 cm stems [4].
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Previous chemical investigations of the plants from the genus Saxifraga led to the isolation of
diverse types of chemical ingredients, including anthocyanins [5], flavonoids [6] [7] [8],
phenolic [9] [10] , glycoside [11] [12],amides [13], steroids [14], alkanes [15], g-
pyronederivatives [16] and diarylheptanoids [17].The essential oils from many Saxifraga
species have been extracted and analyzed using gas chromatography (GC)-based techniques
[18] [19] [20]. Terpenes and aliphatics have been found to be the dominant constituents of the
essential oils. Most of the species are used in traditional medicine and are employed to treat
hepatitis and tumours, influenza, osteoarthritis and purgation [21].
The aims of ours investigation is to valorise the biological potentialities of this species which
has never been evaluated before.
2. METHODS
2.1. Plant material
The Saxifraga veronicifolia Pers., samples were harvestedfrom the mountain of from
the mountain of Megriss (X: 5° 18’ 20” Y: 36° 18' 30” and X’: 5° 24' 7” Y’:36° 21’ 54”) and
determined in the laboratory of National Institute of Agricultural Research – Setif – Algeria
by Dr. Nouioua Wafa , dried in shad and conserved for ulterior use.
2.2. Preparation of methanol extract
The areal parts of Saxifraga veronicifolia was powdered and macerated in 80 %
methanol for 24, 48 and 72 hours, at the laboratory temperature (1:10 w/v, 10 g of dried herb).
Figure 1: Photograph Saxifraga veronicifolia Pers
taken from Megriss setif Algeria 2016
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The extract was collected, filtered and evaporated to dryness under vacuum [22]. The dry
extract was stored at a temperature of -18 °C for later use.
2.3. Determination of Total Phenolic Content
For total polyphenol determination, the Foline Ciocalteu method was used [23]. The
samples (0.2 mL) were mixed with 1 mL of the Folin-Ciocalteu reagent previously diluted
with 10 mL of deionized water. The solutions were allowed to stand for 4 min at 25 °C before
0.2 mL of a saturated sodium carbonate solution (75 mg/mL) was added. The mixed solutions
were allowed to stand for another 120 minutes before the absorbance were measured at 765
nm. Gallic acid was used as a standard for the calibration curve. The total phenolic
compounds content was expressed as mg equivalent of Gallic acid per gram of extract (mg
EAG/GE).
2.4. Determination of total flavonoids content
The flavonoids content in the extract was estimated by the Aluminium chloride
solution according to the method described by Bahorun et al., (1996) [24]. Briefly, 1 mL of
the methanol solution of the extract was added to 1 mL of 2 % AlCl3 in methanol. After 10
minutes, the absorbance was determined at 430 nm. Quercetin was used as a standard. Results
were expressed as mg equivalent Quercetin per gram of extract (mg EQ/GE).
2.5. DPPH Assay
The donation capacity of extract was measured by bleaching of the purple-coloured
solution of 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH) according to the method of Hanato
et al., (1998) [25]. One millilitre of the extract at different concentrations was added to 0.5
mL of DPPH-methanol solution. The mixtures were shaken vigorously and left standing at
the laboratory temperature for 30 minutes in the dark. The absorbance of the resulting
solutions was measured at 517 nm. The antiradical activity was expressed as IC50
(micrograms per millilitre). The ability to scavenge the DPPH radical was calculated using the
following equation:
DPPH scavenging effect (%) = [(A0 – A1)/ A0]X100
Where:
A0: the absorbance of the control at 30 min
A1: is the absorbance of the sample at 30 min. Butylated hydroxytoluene (BHT) was used as
standard [26].
2.6. Reducing power
The reducing power was determined according to the method of Oyaizu (1986) [27].
2,5 mL of the extract were mixed with 2.5 mL of sodium phosphate buffer (pH 6.6 ; 200
mmol/L) and 2.5 mL of potassium ferricyanide (10 mg/mL). The mixtures were incubated at
50 °C for 20 minutes. After cooling, 2.5 mL of trichloroacetic acid (100 mg/mL)
were added; the mixtures were centrifuged at 200g for 10 minutes. The upper layer (5 mL)
was mixed with 5 mL of deionized water and 1 mL of 1 mg/mL ferric chloride, and the
absorbance was measured at 700 nm against a blank. EC50 value (mg extract/mL) is the
effective concentration at which the absorbance was 0.5 for reducing power and was obtained
by interpolation from linear regression analysis. Ascorbic acid was used as a reference
standard [28].
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2.7 Antimicrobial activity:
Bacteria Strains were obtained from the American Type Culture Collection: Gram-
positive bacteria (Staphylococcus aureus ATCC25923 and Bacillus subtilis ATCC6633),
Gram-negative bacteria (Escherichia coliATCC25922 and Proteus mirabilis) and one yeast:
Candida albicans ATCC1024. Muller Hinton agar was used for bacteria culture and
Sabouraud for yeast.
2.7.1 Anti-bacterial Activity
Agar disc diffusion method was employed for the determination of antibacterial activities of
the extract [29] [30]. Briefly, a suspension of the tested microorganism (108 CFU / mL) was
spread on the solid media plates. Filter paper discs (6 mm in diameter) were impregnated with
10 μL (100 mg/mL) of the extract and placed on the inoculated plates. These plates were
incubated at 37 °C for 24 hours. Gentamicin (10 μg/disc) was used as a standard and
dimethylsulfoxide DMSO as a control.
The antibacterial activity was determined by measuring of inhibition zone diameters (mm)
and was evaluated according the parameters suggested by Alves et al. (2000) [31]:
<9 mm, inactive ;
9–12 mm, less active ;
13–18 mm, active;
>18 mm, very active.
2.7.2 Antifungal activity
The antifungal activity was tested by disc diffusion method with modifications [29]. Candida
albicans suspension was obtained in physiological saline 0.9 % from a culture in Sabouraud
(incubated before 24 hours at 37 °C), adjusted to 105 CFU / mL.
One hundred microliter of suspension was placed over agar in Petri dishes and dispersed.
Then, sterile paper discs (6 mm diameter) were placed on agar to load 10 μL (100 mg/mL) of
each sample. Amphotericin 100 µg was used as standard and dimethylsulfoxide DMSO as
control. Inhibition zones were determined after incubation at 27 °C for 48 hours.
2.8. Inhibition of proteins denaturation
The reaction mixture (5 ml) consisted of 0.2 ml of egg albumin (from fresh hen’s egg),
2.8 ml of phosphate-buffered saline (PBS, pH 6.4) and 2 ml of varying concentrations of
extract so that final concentrations became 100, 200, 400, 500 and 800 μg/ml. A similar
volume of double distilled water served as the control. The mixtures were incubated at 37 ±
2ºC for 15 minutes and then heated at 70ºC for 5 minutes. After cooling, the absorbance
was measured at 660 nm by using the vehicle as a blank. Diclofenac sodium was used as the
reference drug [32]. The denaturation of protein inhibition by the extract and standard was
expressed as percentage by using the formula:
Percentage of inhibition = (Control – Test)/Control x 100 [33].
2.9. Statistical analysis
Results were expressed as the mean ± standard deviation. Data was statistically
analysed using t test of Student and Fisher test with the criterion of P values < 0.05 to
determine any significant differences between methanol extract of Saxifraga veronicifolia
Pers and standards, using Graph pad prism 5 Demo Software.
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3. RESULTS AND DISCUSSION
The extraction method gave a yield of 17.27 % contain 17, 36±0, 41 mg EAG/GE of
phenolic compounds and 17.19±0.62 mg EQ/GE of total flavonoids.
Antioxidants are both natural and synthetic compound, able to scavenge free radicals and to
inhibit oxidation processes [34]. The scavenging effect of extract increased with their
concentrations to similar extents (Figure 1). Results revealed that methanol
extract Saxifraga veronicifolia has a low free radical scavenging potential with IC50 of
108,02±17,64 μg/mL*** against 6,29±1,12 μg/mL of free for BHT.
050 100 150 200
0
20
40
60
80
100
BHT
Saxifraga veronicifolia
Concentration ( µg/mL)
Scavenging effects (%)
Figure 1: DPPH scavenging effect of extract of methanol extract Saxifraga veronicifolia
Reducing power of the fractions was assessed using ferric to ferrous reducing activity as
determined spectrophotometrically from the formation of Perl’s Prussian blue colour complex
[35]. The reducing power of crude extract of Saxifraga veronicifolia was compared with
ascorbic acid (Figure 2). The results show an increase in reducing power of the extract as the
extract concentration increases. EC50 of extract was 55.23±0.48 μg/mL***, against the ascorbic
acid 8, 64±0,09μg/mL. Hence, the results concur with those of DPPH and indicates a weak
antioxidant activity of polar molecules.
050 100 150 200
0.0
0.5
1.0
1.5
2.0
Ascorbi c acid
Saxifraga veronicifolia
Concentration ( µg/mL)
optical density
Figure 2: Reducing power activity of methanol extract Saxifraga veronicifolia
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A low antimicrobial activity of extract was registered against the used strains(Tables.1).
Table 1: Antimicrobial activity of standard and methanol extract Saxifraga veronicifolia
Escherichia
coli
Bacillus
subtilis
Staphylococcus
aureus Candida
albicans Proteus
mirabilis
methanol
extract
6,72 9,13 NI NI 9,05
Standards 18,50±0,41 23,83±0,62 18,53±0,41 15,58±0,12 23,83±0,62
Control NI NI NI NI NI
Denaturation of proteins is a well-documented cause of inflammation. Several anti-
inflammatory drugs have shown dose-dependent ability to inhibit thermally-induced protein
denaturation [36]. Protection of proteins from the denaturation induced by heating of extract
reach a maximum of 34,80± 4,25 % at 800 µg/mL against 90,13± 3,71 % of Declofenac at
the same concentration (figure 3).
100 200 400 600 800
0
20
40
60
80
100
Diclofenac Sodium
Saxifraga
veronicifolia
concentration ( µg/ml)
percntage of protein
denaturation inhibition
Figure 3: Protein denaturation inhibition power of standard and methanol
extract Saxifraga veronicifolia
This result represents a moderate anti-inflammatory power of extract.
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4. CONCLUSION
This presents investigation is the first report of valorisation of methanol extract
ofSaxifraga veronicifolia. The In-vitro antioxidant, anti-inflammatory and antimicrobial
activities were low to moderate in comparison of the used standards.
In spite the found results, this species is a better candidate for a further investigations
especially in isolation of molecule domain.
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PhytoChem & BioSub Journal
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ISSN 2170-1768
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... typhimirium, E. coli and S. dysenteriae with inhibition zone diameters ranging from 14 to 21 mm while the most active extract against B. cereus and S. aureus is the total extract (Nouioua and Sofiane, 2019). ...
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The medicinal uses of saffron (Crocus sativus Linnaeus) have a long history beginning in Asian countries since the Late Bronze Age. Recent studies have validated its potential to lower the risk of several diseases. Saffron has been suggested to be effective in the treatment of a wide range of disorders including coronary artery diseases, hypertension, stomach disorders, dysmenorrhea and learning and memory impairments. In addition, different studies have indicated that saffron has anti-inflammatory, anti-atherosclerotic, antidiabetic, antigenotoxic and cytotoxic activities. The anticonvulsant and anti-Alzheimer properties of saffron extract were shown in human and animal studies. The efficacy of Crocus sativus in the treatment of mild to moderate depression was also reported in clinical trial. Many of these medicinal properties of saffron can be attributed to a number of its compounds such as crocetin, crocins and other substances having strong antioxidant and radical scavenger properties against a variety of radical oxygen species and pro-inflammatory cytokines.
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Growing interest to use natural preservatives and spices with antimicrobial effects and large amounts of floral bio-residues (92.6 g per 100 g) generated and wasted in the production of saffron spice guided this study to evaluate the opportunity to expand the uses of C. sativus flowers (petals and stamens), beyond the spice (dried stigmas). The antibacterial potential of total extracts and different sub-fractions of floral bio-residues of saffron production (petals and stamens) were primarily evaluated against five bacterial strains potentially causing food-borne disease (Bacillus cereus, Staphylococcus aureus, Salmonella enterica, Escherichia coli and Shigella dysenteriae) using well diffusion method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were determined by macrodilution method. Methanol extract of petals had shown more antibacterial activity against S. aureus, S. enteric, and S. dysenteriae compared to stigma. Methanol extract and ethyl acetate sub-fraction of stamens showed more antimicrobial effect against B. cereus and E. coli. The petals total extract showed the most antibacterial activity against Shigella dysenteriae (MIC 15.6mg/ml) while the ethyl acetate and chloroform sub fractions showed the maximum effect against Bacillus cereus(MIC 62.5mg/ml). Stamen methanol total extract and aqueous sub fraction have the maximum effect against Staphylococcus aureus and Bacillus cereus (MIC 62.5mg/ml) while the ethyl acetate sub fraction has the best effect against Shigella dysenteriae (MIC 15.6mg/ml). Results showed that both petals and stamens could act as new and natural sources of antibacterial agents with food industrial applications.
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This study reports the in vitro anti-inflammatory activity and in vitro antioxidant activity of a polyherbal extract (PHE). The PHE was prepared from the the following plants viz, Saraca indica, Symplocos racemosa, Hemidesmus indicus, Aloe vera, Asteracantha longifolia, Erythrina indica and Tribulus terrestris. In the in vitro tests for anti-inflammatory activity, the membrane stabilizing activity and thermally induced protein denaturation were studied. The PHE exhibited significant membrane stabilizing property. Thermally induced protein denaturation was significantly inhibited by the PHE. The antioxidant capacity of the PHE was studied in vitro using total antioxidant capacity and reducing power. The results revealed that the PHE showed very potent antioxidant capacity.
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Saxifraga tangutica Engl., is a medicinal herb that grows on the Qinghai-Tibet Plateau. Extracts and phenols from the Qinghai population have been subjected to antioxidative assays against DPPH radical-scavenging and reducing power (FRAP). The 50% ethanol extract showed strong antioxidative activity against DPPH and FRAP, with IC50±SEM [μg/mL] values of 9.38±0.46 and 15.46±0.52, respectively. The antioxidative activity-guided fractionations were performed according to the DPPH and FRAP screening results. Fourteen fractions from the 50% ethanol extract showed dissimilar antioxidative activity against DPPH and FRAP of 8.16±0.76~38.42±0.58μg/mL and 13.22±0.68~61.47±0.49μg/mL. The chemical assay-guided separation of the active fractions (fractions 3, 6, 7 and 8) led to eight phenols: protocatechuic aldehyde (1), ethyl gallate (2), rhododendrin (3), p-hydroxyacetophenone (4), rhododendrol (5), protocatechuic acid ethyl ester (6), frambinone (7) and ethylparaben (8). All phenols are reported here for the first time from S. tangutica Engl. Protocatechuic aldehyde (1), ethyl gallate (2), rhododendrin (3) and protocatechuic acid ethyl ester (6) showed strong antioxidative activities (IC50±SEM [mM] between 8.79±0.15 and 4.25±0.47 and between 6.15±0.48 and 2.83±0.49) against DPPH and FRAP.
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About 68 kg of flowers are needed to produce 1 kg of saffron spice, while 63 kg of bio-residues composed of tepals, stamens and styles are generated. The proximate composition, minerals, dietary fiber (DF), sugars, anions and organic acids of flowers of saffron, their parts and bio-residues from saffron spice production have been analyzed. Whole flowers have high ash (7.39 mg/100 g), protein (10.07 mg/100 g) and available carbohydrates (61.2 mg/100 g), and are low in lipids (3.16 mg/100 g). Stamens are the flower parts with the highest contents of ash (11.43 mg/100 g), protein (24.05 mg/100 g), lipids (10.73 mg/100 g), total DF (32.2 mg/100 g) and insoluble DF (21.1 mg/100 g), and the lowest available carbohydrates (33.8 mg/100 g) and total sugars (4.3 mg/100 g). The insoluble/soluble DF ratios of floral bio-residues (1.2), stamens (1.9) and stigmas (1.3) are suitable as a balanced source of DF. These results could contribute to the using flowers of saffron as food, as well as the development of new food products from flowers of saffron and the management and exploitation of the bio-residues obtained in saffron spice production.
Centella asiatica is a valuable medicinal herbaceous aromatic creeper which has been valued for centuries in ayurvedic medicine. Phytochemical analysis of Centella asiatica plant extracts revealed the presence of various biochemical compounds such as alkaloids, flavonoids, glycosides, triterpenoids and saponins etc. Since triterpenoids and flavonoids have remarkable anti inflammatory activity, so our present work aims at evaluating the in vitro anti inflammatory activity of Centella asiatica by HRBC membrane stabilization. The inhibition of hypotonicity induced HRBC membrane lysis was taken as a measure of the anti inflammatory activity. The percentage of membrane stabilisation for methanolic extracts and Diclofenac sodium were done at different concentrations. The maximum membrane stabilization of C. asiatica extracts was found to be 94.97 % at a dose of 2000 µg/ml. Therefore, our studies support the isolation and the use of active constituents from Centella asiatica in treating inflammations.
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The fatty acid composition of seed oil of five Saxifragaceae species has been investigated : Grossularia burejensis, Hydrangea petiolaris, Saxifraga aquatica, Saxifraga moschata, Schizophragma hydrangeoides. These species have not been studied previously. In the first one only, gamma-linolenic acid has been found (12%), as well as stearidonic acid (6%). Other are deprived of these acids, but two species are rich sources of linoleic acid (75%):H. petiolaris and S. hydrangeoides. The results obtained here argue for the division of Saxifragaceae into some veiy close families or subfamilies. Among these, we have found that Ribesaceae or Ribesoideae regroupping the genera Grossularia and Ribes, form interesting sources of gamma-linolenic acid. Seven species of the genus Ribes have already been investigated. Black currant (Ribes nigrum) exhibit the highest content of this fatty acid. It has been shown also that crude and cooked red currant fruits (Ribes rubrum) gave the same seed oil composition.
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Introduction: Ashwagandha (Withania somnifera) is an important medicinal plant in Indian traditional system of medicine and traditionally has been used for several medicinal purposes in India. The present study was conducted to evaluate the anti-inflammatory effect of hydroalcoholic extract of ashwagandha against denaturation of protein in vitro. Methods: The test extract at different concentrations was incubated with egg albumin in controlled experimental conditions and subjected to determination of absorbance and viscosity to assess the anti-inflammatory property. Diclofenac sodium was used as the reference drug. Results: The present results exhibited a concentration dependent inhibition of protein (albumin) denaturation by the ashwagandha extract. The effect of diclofenac sodium was found to be less when compared with the test extract. Conclusion: Form the present findings it can be concluded that ashwagandha possessed marked anti-inflammatory effect against denaturation of protein in vitro. The effect was plausibly due to the alkaloid and withanolide contents of ashwagandha.