Role of Salvia officinalis Silver Nanoparticles in Attenuation Renal Damage in Rabbits Exposed to Methotrexate

Document Type : Original Articles


1 Biotechnology and Environmental Center, University of Al-Fallujah, Fallujah, Al Anbar, Iraq

2 College of Education for Pure Sciences, Department of Biology, University of Anbar, Ramadi, Al Anbar, Iraq


Nanomaterials are now considered in an extensive range of applications in various fields such as biotechnology and biomedicine. The present study aimed to investigate the protective role of Salvia officinalis Silver Nanoparticles (SOSNPs) as an anti-oxidant on nephrotic damage induced by methotrexate (MTX) in adult rabbits. Green silver nanoparticles were synthesized using alcoholic extract of Salvia officinalis (S. Officinalis) leaves and were characterized by UV-spectrophotometry and scanning electron microscope. The mixing of the plant extract of S. Officinalis with silver nitrate solution leads to the change of the reaction mixture color to yellowish within 1 h and dark brown after 8 h. For studying the protective role of SOSNPs, a total of 28 adult Wistar albino rabbits were divided into four groups and treated intramuscularly (twice per week) for 45 days as follows: T1: S. Officinalis (150 mg/kg B.W), T2: SOSNPs (150 mg/kg B.W); T3: MTX (0.25 mg/kg B.W) and SOSNPs (150 mg/kg B.W); T4: MTX (0.25 mg/kg B.W).  Blood was collected at 0, 15, 30, and 45 days using retro-orbital sinus and cardiac puncture technique, and the serum factors including malondialdehyde (MDA), glutathione (GSH) in serum, creatinine, as well as blood urea nitrogen and uric acid concentrations were measured at the next step. The results indicated that MTX (T4) caused a case of oxidative stress by a significant decrease in GSH and MDA as well as an increase in serum creatinine, urea, and uric acid concentrations. On the other hand, the protective roles of S. Officinalis and SOSNPs given concurrently with MTX were clarified in T2 and T3 groups, where there was the alleviation of renal damage through the correction of the previously mentioned parameters as well as the correction of anti-oxidant status. Finally, the present study documented the anti-oxidant activity and renal protective effects of SOSNPs against the damaging effects of MTX in rabbits.


Main Subjects

1. Introduction

Nanoparticles are clusters of atoms ranging between 1-100 nm in size, showing different or improved physicochemical properties depending on their distribution, size, and morphology ( 1 , 2 ). Nowadays, silver nanoparticles (AgNPs) are extensively used in many biological applications ( 3 ) such as drug delivery, treatment approach, diagnosis, medical device, and protocol of health care ( 4 , 5 ). The biological methods for the synthesis of nanoparticles have attracted researchers' attention using the reduction and stabilization of some enzymatic or non-enzymatic agents, including enzymes ( 6 , 7 ), microorganisms ( 8 ), fungus ( 9 ), as well as plant extracts ( 10 ). Green synthesis is preferable to the physical and chemical methods as it is eco-friendly and easily handled with no need for high temperatures, pressure, energy, or toxic chemicals ( 11 , 12 ).

Different plant extracts were used as reducing agents in green methods such as Nigella arvenus ( 13 ), Nigella sativa ( 14 ), Crocus sativus ( 15 ), Alliumm sepa ( 16 ), Lantana camara ( 17 ), Saccharum officinarum ( 18 ), and Salvia officinalis ( 7 ). The AgNPs have an extensive range of antimicrobial ( 19 , 20 ), antiviral, as well as antifungal and anti-inflammatory activities ( 21 , 22 ). Some studies revealed that wound dressing containing AgNPs prompted the healing of leg ulcers in humans, acting as an anti-bacterial and anti-inflammatory agent ( 23 , 24 ). The anti-cancer activity of AgNPs has also been reported in the in vitro and in vivo studies ( 25 - 28 ). The assumption is that the highest anti-oxidant activity of green nanoparticles might be due to the preferential adsorption of anti-oxidant activity from plant extract onto the surface of the nanoparticles ( 19 ). It is noteworthy that AgNPs act as pro-inflammatory at high doses and anti-inflammatory at low doses ( 29 ).

There were few studies ( 30 ) concerning the use of S. Officinalis as a reducing agent in the green synthesis of AgNPs and their systemic applications. In this investigation, alcoholic extract of S. Officinalis leaves was used for green synthesis of AgNPs, and the hepatic and renal protective effects of S. Officinalis and AgNPs were studied in MTX treated rats.

2. Materials and Methods

2.1. Biosynthesis of Silver Nanoparticles Using Salvia officinalis Plant

2.1.1. Collection and Preparation of Ethanolic Extract of Salvia officinalis

The well-dried leaves of the S. Officinalis plant were collected in March 2020 from the Baghdad market in Iraq. After differential diagnosis, an amount of 20 g of S. Officinalis leaves has been powdered and sieved with fine mesh. The ethanol extracts of S. Officinalis were prepared by mixing 10 g of the dry sample with 100 ml of ethanol, shaken well, kept for 24 h, and then filtered by employing a Whatman filter paper No.1 (125 mm pore size). The supernatant was s filtered again using the mentioned filter paper, and heat treatment for the prepared sample was performed at 80°C for concentrating the extract and removing the residual ethanol. Afterward, it was taken and extracted with Soxhlet apparatus using ethanol 70%, and at the next step, the extracted solvent was exposed under reduced pressure in a rotary evaporator until it became completely dry, and then it was kept at 4°C for analysis ( 31 ).

2.1.2. Synthesis of Phyto-Silver Nanoparticles

An amount of 1500 mg of leaf extracts of S. Officinalis plant was added to 10 ml of 1 mM aqueous silver nitrate solution and kept at room temperature for 8 h to produce AgNPs. The changes in the solution color were measured every 1 h and for 8 h. The changes in the color intensity were noticed after reducing Ag+ to AgNPs by leaf extracts of S. Officinalis plants with increasing reaction time.

2.2. Separation and Identification of Silver Nanoparticles

2.2.1. Spectrophotometry

The optical absorbance of the synthesized AgNPs was performed using a UV-visible spectrophotometer (Perkin-Elmer lambda 750 spectrophotometers) between the wavelengths of 300 and 800 nm at a resolution of 1 nm. The reaction mixture was diluted five times with distilled water and used for UV-visible analysis. The absorbance of this solution was measured at a one-hour interval for 1-8 h, and the changes in the color were gradually observed as it turned dark brown at the end of 8 h ( 32 ).

2.2.2. Centrifugation

Silver nanoparticles solution was centrifuged at 10,000 rpm for 30 min. The pellets were washed three times with 20 ml of distilled water and finally dried at 60°C to dispose of the free proteins/catalysts that are not topping the AgNPs. The centrifugation process was performed at different time intervals of 1-8 h to recognize the effect of time incubation on the amount of synthesized AgNPs ( 33 ). After the centrifugation of AgNPs solution at a different incubation time interval (1-8 h), the samples were washed and weighed three times with 20 ml of non-ionized water to remove free proteins/enzymes in AgNPs.

2.2.3. Scanning Electron Microscopy

The biomasses had settled in the base of the cone-like carafes, and the suspension was tested for scanning electron microscopy (SEM) perception. The scanning electron microscopy of the aqueous solution samples of AgNPs was prepared by setting one drop of the solution on the carbon-covered copper grids, the films on the SEM system were permitted to stand for two min after, and the extra solution was then removed using a blotting paper and drying the grid. The size appropriation of the subsequent nanoparticles was evaluated based on SEM micrographs ( 34 ).

2.3. Animals and Housing

The present study was conducted on 28 adult Wistar albino rabbits (aged three months and weighted 1000±100 g), which were adopted after two-week acclimatization in the College of Veterinary Medicine, Baghdad University Iraq, from March 2020 to August 2020. Moreover, they were housed in well-ventilated rooms inside plastic cages (five rabbits/cage) and fed on a standard pellet diet and drinking water ad libitum during the experiment. The room temperature was kept at 23±2ºC and ventilated for 12 h. In addition, the light/dark cycle with a light on from 06:00 p.m. to 06:00 a.m. was provided along the experiment period.

2.4. Experimental Design

A total of 28 adult rabbits were randomly selected, equally divided into four T1, T2, T3, and T4 groups, and treated (twice per week) intramuscularly for 45 days as follows: T1: animals received S. Officinalis (150 mg/kg B.W) by intramuscular injection; T2: animals received SOSNPs (150 mg/kg B.W); T3: animals were injected both MTX (0.25 mg/kg B.W) and SOSNPs (150mg/kg B.W) intramuscularly; T4: animals received MTX (0.25 mg/kg B.W). The rabbits of all groups were considered the control group at day zero and injected only doubled distill water intramuscularly.

Blood samples were collected at day zero and every 15 days of the experiment, and at the end of the experiment, blood samples were collected via retro-orbital sinus technique and cardiac puncture technique. At the same time, the rabbits were anesthetized by intramuscular injection of xylazine (40 mg/kg B.W) and ketamine (90 mg/kg B.W) using a disposable syringe. Moreover, blood samples were kept in tubes (non-heparinized) and let for 30 min for standing, then serum was gained by centrifugation for 15 min at 3000 rpm, and tightly stopper tubes were kept frozen at -20°C for following chemical analysis; then, it was used for measuring serum concentrations of MDA (Merck, Germany) ( 35 ), GSH (Cypress Diagnostics-Belgium Kit), creatinine and uric acid (Cypress Diagnostics-Belgium Kit), and urea (HCUSABIO-China Kit) ( 36 ).

2.5. Statistical Analysis

Data have been analyzed statistically using Statistical Package for the Social Science (SPSS) software (version 20). Moreover, statistical analysis of data was conducted based on Two-Way Analysis of Variance (ANOVA) using a significant level of P<0.05 and specific group differences were determined using Least Significant Differences (LSD) as presented by ( 37 ).

3. Results and Discussion

3.1. Synthesis and Identification of Silver Nanoparticles

The biogenic synthesis of AgNPs using S. Officinalis leaf extracts was carried out in the present investigation. The mixing of the plant extract of S. Officinalis with silver nitrate solution (1 mM) led to changing of the reaction mixture color to yellowish within 1 h and also dark brown after 8 h (Figure 1), indicating the generation of AgNPs, due to the reduction of silver metal ions (Ag+) into AgNPs via the active molecules present in the S. Officinalis plant extracts.

Figure 1. Changes in color after the reduction of Ag+ to AgNPs by S. Officinalis plant leaf extract at 1-8 h

The formation of AgNPs was monitored with color change and a UV-Vis spectrophotometer. The color of the reaction mixture started to change to yellowish within 1 h and to dark brown color after 8 h. The absorption spectra of AgNPs solution consist of a single sharp surface plasmon resonance (SPR) band at 400 nm (Figure 2 and Table 1). Moreover, the most characteristic part of the silver solution is a narrow plasmon absorption band observable in the 350–600 nm regions, and the distinct visible peak was observed at a wavelength of 430 nm.

Figure 2. A diagram of the absorbent rates of SOSNPs at different times

Duration (h) 1 h 2 h 3 h 4 hr5 h 6 h 7 h 8 h
Absorbance at 434nm (peak) 0.1566 0.1930 0.2430 0.272 0.3323 0.4018 0.4300 0.4587
Table 1. Values of absorbance obtained by a spectrophotometer at a wavelength of 434nm at different times

The changes in pellet weight (mg/time) indicated a correlation between incubation time and the amount of synthesized AgNPs, presented in table 2. After 1 h and 2 h of incubation times, there was a decrease in pellet weight compared to 6 h, 7 h, and 8h of incubation times. The amount of 1 ml of S. Officinalis plant extract mixing with 50 ml of 1 mM AgNO3 solution provided a deep dark color pellet (Figure 1).

Time (h) 1 h 2 h 3 h 4 h 5 h 6 h 7 h 8 h
SOSNPs (g) 0.0149 0.185 0.248 0.265 0.362 0.422 0.438 0.457
QTY (ml) 50 50 50 50 50 50 50 50
Table 2. Amounts of AgNPs synthesized by S. Officinalis extract

Scanning electron microscopy images of AgNPs solution are shown in (Figure 3), which indicate the adsorption and/or deposition of AgNPs onto the surface of roughly sphere-shaped polydispersed particles. The silver nanoparticles that emerged in the images have a variety of shapes such as spherical, triangle, and irregular. As shown in figure 4, the presence of ring patterns in the selected area of electron diffraction reveals the single face-centered cubic crystalline nature of the spherical nanoparticles with a preferential growth direction along the Ag+. The shape evolution of protomorphic AgNPs was indicated in electron microscope images of samples prepared at different times ( 38 ). Moreover, the average size of AgNPs was between 20-50 nm, with a few larger particles exceeding 80 nm only in the case of S. Officinalis leaf extract at the longer reaction times (Figure 4).

Figure 3. Crystalline clusters of AgNPs obtained by scanning electron microscopy (2000X)

Figure 4. Crystalline clusters of AgNPs obtained by scanning electron microscopy (4000X)

The present study provided evidence that the S. Officinalis leaves and flowers were excellent sources for synthesizing stable SOSNPs in shorter times. The color change was due to the reduction of silver ions, indicating the formation of AgNPs. The mechanism by which the SOSNPs could be synthesized by plant extract may be explained by the high total content of phenols and flavonoids ( 39 , 40 ). The mechanism with high reduction capacity ( 41 , 42 ).

These nanoparticles exhibited yellowish-brown color in an aqueous solution due to the excitation of surface plasmon vibrations in AgNPs, which is consistent with the results of some previous investigations ( 8 , 43 ). The S. Officinalis leaf appears with organic compounds such as tannins, terpenoids, steroids, glycosides, and benzenoids present ( 44 ), which are responsible for the efficient stabilization of nanoparticles and reduction of metal ions.

3.2. Separation of Salvia officinalis Silver Nanoparticles

It is renowned that AgNPs exhibit dark brown color, depending on the intensity and the size of nanoparticles; the colors appear due to the excitation of SPR of AgNPs ( 18 ). The spectra absorption of AgNPs solution consists of a single sharp SPR band at 400 nm; this result is consistent with the findings of some other studies ( 45 - 47 ).

It was observed from the spectra that the SPR peak of AgNPs occurs at 434 nm with a high absorbent; this peak is due to the typical SPR of AgNPs, indicating that the particles were well-dispersed without aggregation and it was extremely specific for AgNPs. These findings are in line with some previous studies ( 48 - 50 ), in which, reduction of silver was indicated as well, showing that using the UV method, the reduction of silver is faster than that using the conventional method.

The SEM image was taken after seven days following the completion of all reactions; even after seven days, the particles did not accumulate. This observation confirmed that S. Officinalis extract might act both as a reducing and a stabilizing agent in synthesizing SOSNPs ( 51 ).

3.3. Effects of Salvia officinalis and Salvia officinalis Silver Nanoparticles on Control and Methotrexate Treated Rabbits

During the experimental period, a significant increase (P<0.05) in the serum GSH was observed in the T2 and T3 groups after intramuscular injection of green nanoparticles alone or concurrently with MTX compared to the value in T4 and T1 groups. Highest significant (P<0.05) increase in GSH was observed in group T2 at the end of the experiment in comparison with the value in other treated groups (Table 3). The results also indicated a significant (P<0.05) decrease in serum MDA concentration in T1, T2, and T3 groups compared to the value in MTX treated (T4) group along the experimental period. A type of green nanoparticles (i.e., SOSNPs) and MTX-AgNPs groups indicated the highest significant (P<0.05) decrease in serum MDA concentration (T2 and T3 groups) compared to the value in other treated groups (Table 4).

Treatment Time T1 T2 T3 T4
0 120.00±2.16a 119.00±4.03b 117.20±4.41b 120.00±2.02a
15 123.40±2.13a 155.10±4.1a 139.10±2.75a 103.11±4.6b
30 118.60±1.92a 154.30±6.70a 139.10±4.70a 111.60±3.32ab
45 124.20±1.22a 155.30±1.2a 146.30±0.93a 100.10±2.98b
Table 3. Effects of S. Officinalis and SOSNPs on reduced GSH concentration (μmol/L) in normal and MTX treated rabbits
Treatment Time T1 T2 T3 T4
0 346.00±4.80a 337.00±8.06a 354.00±11.2a 360.00±5.14b
15 308.00±5.07a 259.00±4.77b 244.00±6.08b 415.00±23.1a
30 340.00±10.6a 298.00±6.17b 228.00±7.46b 418.00±17.0a
45 333.00±11.11a 267.00±10.12b 266.00±13.6b 440.00±17.3a
Table 4. Effects of S. Officinalis and SOSNPs on MDA concentration (μmol/L) in normal and MTX treated rabbits

A significant decrease in serum MDA and a significant increase in GSH were observed in S. Officinalis treated group. The anti-oxidant activity of S. Officinalis was documented by many investigations ( 52 , 53 ), which attributed to their flavonoid content with their free radical scavenging activity. This result is also in line with ( 54 ), where S. Officinalis concentration in rabbits (0.3-13.33 mg/ml in water) has anti-oxidant protection through raising the GSH content and increasing the glutathione peroxidase (GPx) activity. Many phytocompounds, such as polyphenols, flavonoids, tannin, sugars, alkaloids, as well as triterpenoids/steroids, have been reported to be present in the S. Officinalis, which are responsible for potent anti-oxidant, anti-inflammatory, larvicidal, and other medicinal properties ( 44 ).

The injection of SOSNPs to rabbits caused a correction in the disturbance of anti-oxidant status (increase in MDA and decrease in GSH) caused by MTX. The free radical scavenging potential of AgNPs is documented in ( 7 , 16 , 19 ), and its observed strong anti-oxidant properties have been evaluated to be 98.6% (55). An increase in the activities and mRNA expression levels of glutathione peroxidase (GSH-pX) and glutathione reductase (GSH-RD), superoxide dismutase (SOD), catalase (CAT), as well as total anti-oxidant capacity with a significant decrease in MDA in normal compared to diabetic rabbits were obtained after exposure to 10 mg/kg B.W of AgNPs ( 56 ). Anti-oxidant enzymatic activities were enhanced, and a cell protection mechanism was provided against membrane damage induced by oxidative stress by catalyzing peroxide elimination ( 57 ). It has been assumed that the highest anti-oxidant activity of nanoparticles might be due to preferential adsorption of anti-oxidant material of plant extracts on the surface of nanoparticles ( 19 ). It is noteworthy that large-size AgNPs are more effective anti-oxidants than smaller ones ( 58 - 60 ).

A significant decrease in GSH and a significant increase in MDA were observed in MTX treated group, indicating oxidative stress. Nuclear factor-erythroid 2 related factor 2 (Nrf2) is a transcriptional activator that can act as a sensor for oxidative stress and has an important role in the regulation of defensive genes activation and induction of anti-oxidant enzymes as SOD, CAT, and GPx that participated in the suppression of injury evoked by reactive oxygen species ROS and protection of cells against oxidative stress injurious effects ( 61 , 62 ). It has been found that MTX induced a decrease in mRNA of Nrf2 as well as

Nrf2 binding capacity, which can partially explain the reduction of the anti-oxidant status in MTX treated mice ( 63 ).

3.4. Effects of Salvia officinalis and Salvia officinalis Silver Nanoparticles on Serum Kidney Function in Control and Methotrexate Treated Rabbits

A significant decrease (P<0.05) in serum urea concentration was observed in the T1, T2, as well as T3 groups after intramuscular injection of S. Officinalis, green nanoparticles, and MTX, throughout the experimental period compared to the values in group T4, in which MTX was given (Table 5). It was demonstrated in table 6 that intramuscular injection of S. Officinalis (T1) or green nanoparticles alone (T2), in combination with MTX (T3), caused a significant decrease in serum uric acid concentration in the T1, T2, and T3 groups during the experimental period compared to the values in group T4 (P<0.05). With exception to the value in T4, which demonstrated significant differences in creatinine concentrations, non-significant differences were observed over time in other experimental groups during the experimental period compared to zero time (P>0.05) (Table 7).

Treatment Time T1 T2 T3 T4
0 45.00±0.54a 47.60±1.50a 48.210±3.51a 45.00±1.62c
15 43.20±1.83a 43.20±1.43ab 46.60±1.80a 52.00±2.14b
30 45.30±1.4a 38.40±0.80b 43.10±1.44a 66.10±3.00a
45 44.40±1.86a 43.10±1.80ab 41.30±1.03b 60.00±2.22b
Table 5. Effects of Salvia Officinal's (SO) and Salvia Officinal's Silver Nanoparticles (SOSNP) on concentration (mg/dl) in normal and methotrexate (MTX) treated rabbits
Treatment Time T1 T2 T3 T4
0 0.83±0.05a 0.85±0.04a 0.73±0.04a 0.81±0.05b
15 0.74±0.04b 0.74±0.04b 0.71±0.05a 0.91±0.04a
30 0.72±0.03c 0.73±0.06b 0.71±0.05a 0.93±0.04a
45 0.84±0.06a 0.74±0.05ab 0.68±0.06a 0.93±0.03a
Table 6. Effects of Salvia Officinal's (SO) and Salvia Officinal's Silver Nanoparticles (SOSNP) on serum acid concentration (mg/dl) in normal and methotrexate (MTX) treated rabbits
Treatment Time T1 T2 T3 T4
0 1.55±0.07a 1.57±0.09a 1.70±0.07a 1.45±0.04c
15 1.72±0.06a 1.34±0.07a 1.55±0.06a 3.21±0.05b
30 1.83±0.13a 1.42±0.09a 1.50±0.04a 3.115±0.08ab
45 1.77±0.03a 1.49±0.10a 1.69±0.09a 3.15±0.17a
Table 7. Effects of Salvia Officinal's SO) and Salvia Officinal's Silver Nanoparticles (SOSNP) on serum uric Creatinine (mg/dl) in normal and methotrexate (MTX) treated rabbits

It has been found that small size and high dose of AgNPs caused renal damage by increasing the concentrations of serum creatinine, urea, and uric acid ( 64 , 65 ); however, a low dose of AgNPs (4.8 mg/kg B.W), injected intramuscularly for 28 days in rats, caused renal protective activity; this finding was also demonstrated by ( 60 , 66 ), in which the safeness of in vivo use of low doses of AgNPs with large sizes was recorded. The results also indicated significant variations in renal function tests that alter MTX exposure, indicating the renal toxicity; this finding was also documented by ( 67 - 69 ). It is believed that MTX induces renal injury, either by precipitation in, or a direct toxic effect on the renal tubules. Methotrexate and its metabolites are poorly soluble in acidic environments, promoting potential precipitation in acidic urine ( 70 , 71 ).

A significant decrease in serum urea, creatinine, and uric acid levels was observed in rabbits exposed to SOSNPs, which indicates its re-protection. The high affinity of metal nanoparticles towards nitrogen-containing compounds and their capability to bind them ( 72 ) could be the reason behind the decrease in the concentration of renal biomarkers after treatment of SOSNPs. Moreover, metal nanoparticle surface properties provide some functionalities with small organic molecules, including urea, uric acid, and creatinine, by their various functional groups ( 73 ), which lead to the reduction in their concentration.

Authors' Contribution

Study concept and design: M. A. S. and N. A. S.

Acquisition of data: M. A. S. and N. A. S.

Analysis and interpretation of data: M. A. H.

Drafting of the manuscript: L. F. G.

Critical revision of the manuscript for important intellectual content: M. A. S. and N. A. S.

Statistical analysis: M. A. S. and N. A. S.

Administrative, technical, and material support: M. A. S. and N. A. S.


All the procedures were approved by the ethics committee of the College of Veterinary Medicine, Baghdad University Iraq (No.: 2020-4789-12).

Conflict of Interest

The authors declare that they have no conflict of interest.


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