Utility of silver nanoparticles for the analysis of diosmin and rutin in Persicaria salicifolia extract, authentic and pharmaceutical dosage forms monitored with their haemostatic activity

The present work was conducted to evaluate a simple effective technique for spectrophotometric determination of two flavonoids namely diosmin (1) and rutin (2) in the isolated fractions obtained from Persicaria salicifolia, authentic and pharmaceutical dosage forms, the method is based on the reduction of Ag + cations to silver nanoparticles (Ag-NPs) by the effect of these flavonoids in the presence of polyvinylpyrrolidone (PVP) as a stabilizing agent which result in production of intense brown colour corresponding to the formation of the surface plasmon resonance of produced silver nanoparticles. The plasmon absorbance of the Ag-NPs allows quantitative spectrophotometric determination of these flavonoids where calibration curves derived from the changes in absorbance at λ=415 and 432 nm were linear with concentration range of diosmin and rutin 1-8 and 0.5-7.0 μg/mL while their detection limits (3σ) were 0.26 and 0.12, respectively. The method was applied successfully for the determination of flavonoids in extract, authentic and pharmaceutical dosage forms. The haemostatic activity of the total methanol extract, isolated fractions, authentic and dosage forms were investigated for their usual and silver nanoparticulated forms using tail bleeding time and amount of bleeding assays in rats, where the nanoparticulated samples exhibited more significant activities.

Nanoparticles made of noble metals have been the focus of research for many decades as a result of their intriguing optical properties; among the noble metal nanoparticles, silver nanoparticle gained special interest because of their advantageous properties concerning stability and conductivity (Popescu et al., 2010;Baruwati et al., 2009), in addition, they have been the subject of intensive research because of their unique and tunable surface plasmon resonance (SPR) properties when dispersed in liquid media exhibiting a strong characteristic UV-Vis extinction band that is not present in that of the bulk metal.This extinction band results when the incident photon frequency is resonant with the collective excitation of the conduction electrons and is known as the surface plasmon resonance (SPR).SPR excitation results in wavelength-selective absorption with extremely large molar extinction coefficients where the peak wavelength and intensity of the SPR spectrum is dependent upon the size, shape and inter-particle spacing of the nanoparticle as well as its own dielectric properties and those of its local environment including the substrate, solvent and adsorbates (Baruwati et al., 2009;Nezhad et al., 2010).Silver nanoparticles exhibit significant antibacterial, antiviral, antifungal, antiinflammatory effects, in addition; they are used in cancer diagnosis and treatment (Ahmed et al., 2003).
The present study was conducted targeting bio-green synthesis of silver nanoparticles of the crude extract of P. salicifolia seeds (Figure 2), fractionated diosmin and rutin, their authentic and dosage forms; their characterization, pharmaceutical analysis utilizing very simple and sensitive colourimetric technique relying on their properties as active reducing agents participating in generation of silver nanoparticles with monitoring of their haemostatic activity.

Plant material
P. salicifolia plants were collected in November 2015 from the Nile river irrigation canals, pond edges and irrigation canal bank of Ekhnaway, Tanta, El-Gharbia Governorate, a voucher specimen (C.S. # 0909) was deposited in a herbarium in Pharmacognosy Department, Faculty of Pharmacy, Al Azhar University, Cairo, Egypt.The plant seeds were separated, air-dried, powdered (2 mm mesh) and kept in tightly closed amber coloured glass containers protected from light at low temperature.

Pharmaceutical preparations
Dioven ® tablets containing 500 mg diosmin per tablet with Batch No.1147016 (B) was obtained from Amriya for Pharmaceutical Industries, Alexandria, Egypt and Ruta C 60 ® tablets containing 60 mg rutin per tablet with Batch No. 31304 was obtained from Kahira Pharmaceuticals and Chemical Industries Co., Cairo, Egypt).

Standard solutions
Solutions of 100 µg/mL of diosmin and rutin were prepared by dissolving 10 mg of the pure drug in bi-distilled water.

Animals
Adult albino rats (150-200 g) of either sex were purchased from The Animal House Laboratory, National Research Center, Cairo, Egypt, according to the protocol of the Institutional Animal Care and Use Committee (IACUC) and they were housed in an environmentally control room, maintained at uniform light and temperature conditions of and provided with food and water ad libitum.

Test material
Extract and fractions; total methanol extract, fractionated diosmin, fractionated rutin; authentic material; diosmin, rutin and their pharmaceutical dosage forms, all were tested in their ordinary and nanoparticulated forms.

Extraction and fractionation
300 g of powdered P. salicifolia seeds were exhaustively extracted using soxhlet with 1000 mL of 80% (v/v) aqueous methanol for 1 h to yield the methanol extract which was subsequently filtered under vacuum through Whatmann No. 1 filter paper.The residue was reextracted following the same procedure two more times; the filtered extracts were pooled together and concentrated under vacuum at 40°C to dryness to yield the collective methanol extract (52 g).45 g of the methanol extract were suspended in sufficient volume of distilled water and successively fractionated using n-hexane, methylene chloride and ethyl acetate where each fraction was concentrated under reduced pressure at 40°C; they yielded 7.5 and 11 g, respectively.8 g of the ethyl acetate fraction were chromatographed on 200 g silica gel packed on glass column (3 cm diameter, 0.75 m length), elution with gradient mode was carried out starting water 100% to methanol 100% at flow rate 10 mL min -1 to give 32 fractions; 100 mL each.The collected fractions were monitored with paper chromatography using S1; n-butanol:acetic acid:water (4:1:5 v/v), upper layer, S2; acetic acid:water (15:85 v/v) as solvent systems; the resultant spots were visualized with ferric chloride (Markham, 1982) and aluminum chloride (Stahl, 1969) spray reagents for phenolic compounds and flavonoids, respectively, where similar fractions were pooled together.

Formation of silver nanoparticles
P. salicifolia methanol extract, fractionated diosmin and rutin, authentic diosmin and rutin as well as their pharmaceutical dosage forms were used to produce their silver nanoparticles as follows: in 5 mL volumetric flask appropriate equal volumes of AgNO3 solution (0.1%) and 1% aqueous solution of each of the studied drugs were mixed, then PVP and appropriate amounts of NaOH were added, mixed and completed to 5 mL with bi-distilled water, heated in the water bath at 90 o C for appropriate times to allow the reduction processes to occur which were noticed by the colour change to brownish indicating the formation of the nanoparticles.The brown colour of the produced AgNPs is due to excitation of their surface plasmon resonance; this was observed first by naked eye and subsequently subjected to spectroscopic characterization.The obtained nanoparticles were purified through centrifugation at 10,000 rpm for 5 min, washed and dried in vacuum chamber for 24 h at 35°C, absorbance was measured at the suitable wavelength against reagent blank treated similarly (Praba et al., 2015).

Characterization of silver nanoparticles
The shape, size and morphology of the silver nanoparticles were determined using Scanning Electronic Microscopy (SEM) and Transmission Electron Microscopy (TEM).

Assay of tablets
For either Dioven ® tablets or Ruta C 60 ® tablets, ten tablets were weighed, coat removed and pulverized into fine powder, specific quantity of powdered drugs equivalent to 10 mg pure drug were dissolved in distilled water, solutions were filtered and diluted to 100 mL with distilled water then further diluted to 10 µg/mL.Procedures were completed as in general procedures.

Experimental design for biological studies
One hundred and eighty rats of both sexes were divided into 15 groups; 12 rats each received various treatments [distilled water and tested drugs (10 mg kg

Bleeding time
Bleeding times were determined as follows; each rat was placed in a standard plastic restraining cage with the tail hanging out freely, bleeding time was assessed by cutting the tip of the tail with a sharp pair of surgical scissors (2-3 mm), the tail was placed in an isotonic saline solution with pH 7.4 maintained at 37 ºC immediately after the cut was inflicted.Bleeding time was calculated through a stopwatch was started simultaneously with the immersion of the tail in the saline solution where the time taken was the time from appearance of the first drop of blood to the time when the bleeding stopped completely (Rajasekaran et al., 2010).

Amount of bleeding
The quantity of bleeding was measured as follows; rats were placed individually in a standard plastic restraining cage with the tail allowed to hang out freely, the tip of the tail of each rat was cut (4-5 mm) with a sharp pair of scissors and a stopwatch started immediately, a pre-weighed blotting paper was used to collect all drops of blood that flowed from the site of the inflicted injury, the blotting paper was re-weighed after the appearance of the last drop of blood, the difference in weight of the dry and wet blotting paper was taken as the amount of bleeding (Cipil et al., 2009;Yalcinkaya et al., 2011).

Statistical analysis
Results were expressed as means ± SEM, significance was determined using students t-test and paired mode where results were regarded as significant at P≤0.001 and P≤0.0001 (Elliott and Woodward, 2007).

RESULTS AND DISCUSSION
Fractionation and purification of ethyl acetate fraction of P. salicifolia seeds was carried out resulting in isolation of two major flavonoids and the data gained was as follows: Compound   published spectral data and co-chromatography with authentic sample (Mabry et al., 1970;Agrawal, 1989).
Concerning the preparation of silver nanoparticles in this study, an aqueous AgNO 3 solution including polyvinylpyrrolidone (PVP), as stabilizer, in an alkaline medium was used, then diosmin and rutin were added acting as effective reducing agents for the reduction of silver metal salt (Ag + ) to the Ag-NPs manifested by the brown colour produced upon completion of the reaction (Figure 3).The absorbance of reducing agents exhibited no absorbing peak in visible region (380-700 nm) meanwhile, upon addition of the cited drugs which act as reducing agent, silver ions were reduced to silver nanoparticles and then the absorbance characteristic to the plasmon of the Ag-NPs was observed (415 and 432 nm) for diosmin and rutin, respectively (Table 1, Figures 4 and 5).

Effect of NaOH concentration
The influence of pH on Ag + reduction by the cited drugs is expected since they have a hydroxyphenyl group which can lose H + during oxidation and O-quinone formation process.Because buffered condition failed to obtain silver nanoparticles, NaOH was added to provide enough alkalinity.By addition of NaOH, absorbance increases up to a known concentration of NaOH then decreases the formation of black precipitate which might be due to the Ag 2 O formation.Thus, 1 mL of 0.0025 M NaOH was selected for each diosmin and rutin (Figure 6).

Effect of Silver nitrate concentration
Maximum absorbance values were obtained using 0.7 and 1.0 mL of 0.02 M silver nitrate for diosmin and rutin, respectively (Figure 7).

Effect of Stabilizer type and concentration
An important issue in the preparation of metal nanoparticles is the choice of the capping agent used to protect or stabilize the nanoparticle colloidal metals from agglomeration.Size and morphologies of nanoparticles depends significantly on capping materials.Nanoparticles stabilization is achieved according to the two basic modes: electrostatic and steric stabilization (Januja et al., 2009).Electrostatic stabilization is caused by the columbic repulsion between particles, caused by the electrical double layer formed by ions adsorbed at the particle surface (for example sodium citrate) and the corresponding counter ions.Steric stabilization is achieved because of the coordination of steric ally demanding organic molecules and polymers that act as protective shields on the metallic surface (for example PVP).In this study, PVP and sodium citrate were selected as the stabilizer for preventing of silver nanoparticles agglomeration in which the PVP was better Abs.used compared to sodium citrate.0.7 and 0.5 mL of 0.14 % PVP were optimum for diosmin and rutin, respectively (Figure 8).

Effect of temperature and time of heating
Heating in water bath at 90°C for 40 and 30 min were  sufficient to produce maximum color intensities for diosmin and rutin, respectively (Figure 9).

Linearity
Under the described experimental conditions, standard calibration curves with good linearity for silver nanoparticles formed using authentic diosmin and rutin were constructed by plotting absorbance against concentration (Figures 10 and 11).
A linear correlation was found, the concentration ranges, correlation coefficient, intercept and slope for the calibration curve were calculated, also relative standard deviation, detection and quantification limits were calculated for authentic and fractionated diosmin and rutin (Tables 2 to 4).The validity of the proposed method was assessed by its application to the determination of the studied drugs in their pharmaceutical preparations (Tables 5 and 6).
Student's t-test and F-test (at 95% confidence level) were applied to the results obtained compared with that obtained from reported methods (Moldovan et al., 2010;Younes et al., 2014); the results showed that there were  no significant differences between the proposed and reported methods.Results of different statistical treatment of the data (Table 7).

Accuracy and precision
Accuracy and precision were carried out by six determinations at two different concentrations of the two drugs in the same day (intra-day), and in six different days (inter-day).Percentage relative standard deviation (R.S.D. %) was used as precision.The results of accuracy and precision show that the proposed methods have good repeatability and reproducibility (Table 8).

Nanoparticle characterization
SEM analysis was employed to determine the surface morphology and the topography of synthesized silver nanoparticles where the size of silver nanoparticles ranged from 28 to 33 nm, with an average size 30 nm.The gained SEM images showed that the gained silver nanoparticles were mostly spherical in shape while TEM analysis revealed that most particles were obviously spherical in shape and well dispersed, with an average    size around 30 nm (Figure 12).

Haemostatic activity
The total extract of P. salicifolia seeds significantly (P≤0.001)decreased bleeding time and amount of bleeding when administered orally to rats at doses of 10 mg/kg while the rest of cited drugs exhibited more significant hemostatic activity (P≤0.0001)(Table 9).The percent reduction in the bleeding time of the authentic diosmin, authentic rutin, total extract, fractionated diosmin, fractionated rutin, diosmin dosage form and rutin dosage were, 12.08 to 36.06 and 19.09 to 42.32% for the cited drugs and their nanoparticulated forms respectively while their percent reduction in amount of bleeding were as follows: 12.30 to 39.04 and 16.00 to 46.15%.

Conclusion
The gained results supported the advantage of the use of PVP-craped AgNPs as colorimetric probe for determination of diosmin and rutin in P. salicifolia seeds'  extract, fractions, authentic and dosage form.The method used is economic, simple, rapid, does not require various elaborate treatment and environmentally safe, moreover, this method was successfully validated and exhibited great potential for determination of different samples with high accuracy and precision meanwhile in vivo haemostatic studies revealed that the extract and analyzed drugs exhibited significant haemostatic activity Table 9.Effect of normal control, authentic diosmin, authentic rutin, total extract, fractionated diosmin, fractionated rutin, diosmin dosage form and rutin dosage form and their silver nanoparticulated forms on bleeding time and amount of bleeding in rats.which was manifested as deceased bleeding time and amount of bleeding when orally administered to rats.

Figure 4 .
Figure 4. Absorbance spectra of the silver nanoparticles formed in the presence of 5 µg/mL Diosmin.

Figure 5 .
Figure 5. Absorbance spectra of the silver nanoparticles formed in the presence of 6 µg/mL Rutin.

Figure 6 .
Figure 6.Effect of NaOH on the absorbance spectra of the reaction product between AgNPs and the studied drugs in the presence of PVP.

Figure 7 .
Figure 7. Effect of AgNPs concentration on the absorbance spectra of the reaction product between AgNPs and the studied drugs in the presence of PVP.

Figure 8 .
Figure 8.Effect of PVP concentration on the absorption spectra of AgNPs with studied drugs.

Figure 9 .
Figure 9.Effect of reaction time on the absorption spectra of the corresponding reaction of the studied drugs.

Table 1 .
Analytical parameters for determination of diosmin and rutin through silver nanoparticles formation.

Table 3 .
Determination of authentic diosmin and rutin through silver nanoparticles formation.
*Mean of three different experiments.

Table 4 .
Determination of fractionated diosmin and rutin through silver nanoparticles formation.
*Mean of three different experiments.

Table 5 .
Determination of diosmin and rutin in their pharmaceutical formulations.
*Mean of three different experiments.

Table 6 .
Application of standard addition technique for determination diosmin and rutin in their pharmaceutical formulations.
*Mean of three different experiments.

Table 7 .
Statistical data for determination of diosmin and rutin through silver nanoparticles formation.
*Theoretical values of t and F at p =0.05.

Table 8 .
The intra-day and inter-day accuracy and precision data for determination of diosmin and rutin through silver nanoparticles formation.
*Mean of six different experiments.