The combination of nanotechnology and green synthesis has created new possibilities for plant researchers, for the development of various biomedical applications through the synthesis of metallic nanoparticles. They are used for numerous applications such as the catalysis, electronics, optics, environmental and biotechnology [1-2]. The metals such as gold, silver and copper have been used extensively for the synthesis of stable dispersion of nanoparticles, which are highly efficient in fields such as biological labeling, photonics, and surface-enhanced Raman scattering (SERS) detection [3-4]. Silver nanoparticles are also used in the various fields of bio-sensing, imaging, drug delivery, nanodevice fabrication and medicine [5-6]. The green synthesis of nanoparticles from plant extracts has drawn attention for its simple, rapid, stable, inexpensive and viable alternative method to chemical and physical methods. Recently, different parts of plant extracts have been extensively used for the synthesis of silver nanoparticles by using, Andrographis paniculata , Cathranthus roseus , Ficus fruit , Coleus aromaticus leaf  Glycorhiza galbra root  Cassia alata leaf  Indigofera hirsuta leaf  Allamanda carthica latex  synthetic humic substances  poly(N vinyl 2 pyrrolidone PVP, and also seed aqueous extract of Olea europaea. [16-17] Prangos ferulaceae leaves extract, Cynanchum L extract[18-19] have been used for the biosynthesis of metallic nanoparticles of different sizes and also studied their spectral properties and beneficial application.
It is a well-known that the silver ions and silver based compounds are highly toxic in nature to the microorganisms. This toxic nature depends upon the nanosized particles which are specific in size, shape and exhibits different physical and chemical properties [7-9]. Past studies have shown that high concentrations of nontoxic nano-sized silver particles against different Escherichia coli bacteria exhibited good bactericidal activity [10, 16, 20]. Silver nanoparticles also exhibit antimicrobial properties against different bacterial species; current studies have been focusing on the physicochemical properties and concentrations of AgNPs, which ultimately inhibit E.coli , Staphylococcus aureus , B. subtilis and K. mobilis Salmonella, Shigella and Proteus. So the present study deals with the green synthesis of AgNPs using leaf extract of Andrographis serpyllifolia, to study antimicrobial and antioxidant activity.
Andrographis serpyllifolia is commonly known as round leaf Kariyat, Aaku chandrika. This plant is a trailing herb auxiliary plant species which belongs to the Acanthaceae family, and mostly found in the southern India states of Andhra Pradesh, Tamil Nadu, Karnataka and Kerala. In Andhra Pradesh this plant was collected from the Talakona forest region of Chittoor district. A.serpyllifolia whole plant contains alkaloids, flavonoids, steroids, phenols, saponins, terpenoids, anthraquinones, sugar, glycosides, phycobalamin and tannins . The bioactive phyto-constituents of A. serpyllifolia are associated to have important biomedical applications like antibacterial, anti diabetic, anticancer, anti ulcer, and anti inflammatory activities [22-23] It is also reported that inA.serpyllifolia have the presence of important chemical constituents like serpyllin, apigenin 7,4’- dimethyl ether and tectochrysin compounds of high medicinal value [24-25]. Andrographolide (AG) has been reported as one of the potential bioactive component of A. serpyllifolia which is found to be responsible for several pharmacological and clinical activities [ 26-27]. A.serpyllifolia plant is used as traditional Indian herbal medicine for malaria and dysentery. The root extract of this plant is used to cure fever. The plant extract is used in treating wounds and also found effective in the treatment of jaundice. Aqueous and methanolic extracts of this plant showed anti-proliferative and anti-oxidant properties. A. serpyllifolia have excellent antimicrobial activity, found to be effective in the treatment of several microbial infections. [28-30] The root extracts like chloroform extract, methanolic extract exhibited higher anti-inflammatory activity both in vitro and in vivo conditions .
MATERIALS AND METHODS
Synthesis of silver nanoparticles
The Andrographis serpyllifolia plant leaves were collected from the shrubs growing in and around of Thalakona region of Tirupati and Chittoor district of Andhra Pradesh, India in the months of October and November. The leaves were collected and shade dried for a week at room temperature and then made into fine powder with the help of electric blender. The plant leaf extract was prepared, by adding 5g of finely powder leaves in a 500 ml Erlenmeyer flask with 250 ml of Milli Q ultra pure distilled water and the mixture was heated at 700C for 30 min and then filtered through sterile muslin cloth followed by whatmann No.1 filter paper. This filtrate solution was used as source of extract for preparation of silver nanoparticles and was utilized in subsequent procedures. To the 5ml diluted filtrate, 10ml of 0.002(M) AgNO3 was added and the sample was left at room temperature, until the color of solution changed from pale light green color to light brown and subsequently dark brown. The solution containing AgNPs was confirmed by the dark brown color (Fig. 1). So, in the current study the biosynthesis of silver nanoparticles (AgNPs) with Andrographis serpyllifolia (A. serpyllifolia) plant leaves extract was prepared without any external toxic chemicals which is popularly known as “Green synthesis” or “Green Method”
The bio-reduction of pure Ag+ ions done with the leaf extract of Andrographis serpyllifolia was monitored periodically by sampling of the 1ul and the optical absorbance of silver nanoparticles suspended in distilled water was recorded on UV–Vis Spectrophotometer (Nanodrop 8000 UV-Vis spectrometer) in 220–700nm wavelength range. The reaction solutions were carried out at room temperature on spectrophotometer at a resolution of 1 nm. The FT-IR was carried by using Brucker Tensor 27, Particle size and zeta potential measurement experiments were carried out by using a Nanopartica (Horiba Nanopartica SZ-100 instrument) EDX was performed by Oxford Inca Penta FeTX3 EDS instrument attached to Carl Zeiss EVO MA 15 Scanning Electron Microscope (200 kV) machine with a line resolution 2.32 (in Å). These images were taken by drop coating AgNPs on an aluminum foil. Energy Dispersive Absorption Spectroscopy photograph of AgNPs were carried out by the SEM equipment, as mentioned above. In X-Ray diffractometry (XRD) study was conceded to prove the crystalline nature of the AgNPs using Ultima IV X-ray powder diffractometer (Rigaku Ltd, Tokyo, Japan) by means of Cu Kα radiation source (Madras university, Chennai). TEM analysis was carried out, using 200 KV FEI-Tecnai G2 20 S-TWIN High resolution on Transmission Electron Microscope (VIT, Vellore).
The antimicrobial activity of silver nanoparticles was evaluated against Gram positive; Staphylococcus aureus, Lacto bacillus, Enterococcus Gram negative Escherichia coli, by disc diffusion method. The bacterial strains of microorganisms used for the antibacterial activities of silver nanoparticles were obtained from DST-PURSE Centre, S.V. University, Tirupati. All the bacterial strains maintained on nutrient agar and were sub cultured freshly and used for the studies. Bacterial cultures were prepared by transferring a single colony into a tube containing 10 ml nutrient broth and grown overnight at 37 °C. The overnight grown cultures were prepared in nutrient broth (Himedia, gm/L). The individual microorganisms were prepared by spreading 100 μl of culture on the nutrient agar plate with the help of spreader. Sterile discs were prepared by using Whatmann No.1 filter paper. The discs were placed on agar plates and different concentrations of sample of biosynthesized As-AgNPs were added on the disc with the help of micropipette. The plates were incubated at 37°C overnight and observed next day for zone of inhibition (ZIO) was measured and the results photographed and tabulated.
The antioxidant activity of Andrographis serpyllifolia (A. serpyllifolia) plant leaves extract and biosynthesized As-AgNPs were evaluated in vitro by three methods. All the synthesized compounds showed good free radical scavenging activity against 2,2-Diphenyl-1-picrylhydrazyl(DPPH), Nitric oxide(NO) and Hydrogen peroxide (H2O2) radicals. Ascorbic acid is used as a standard control.
DPPH radical scavenging activity
The hydrogen atom or electron donation ability of the compounds was measured from the bleaching of the purple colored methanol solution of (DPPH) . The spectrophotometric assay uses the stable radical DPPH as a reagent. 1 ml of various concentrations of the test compounds (25, 50, 75, and 100 µg/mL) in methanol was added to 4 mL of 0.004% (w/v) methanol solution of DPPH. After a 30 min incubation period at room temperature, the absorbance was measured against blank at 517 nm. The percent of inhibition (%) of free radical production from DPPH was calculated by the following equation. All the experiments were conducted and radical scavenging activity values were calculated from the absorbance values using the following formula.
Radical Scavenging Activity
Where AC is the absorbance of the control reaction (containing all reagents except the test compound) and AS is the absorbance of the test compound. Tests were carried in triplicate.
Nitric oxide scavenging activity
Nitric oxide scavenging activity was measured by slightly modified methods . Nitric oxide radicals (NO) were generated from sodium nitroprusside. 1 mL of sodium nitroprusside (10 mM) and 1.5 ml of phosphate buffer saline (0.2 M, pH 7.4) were added to different concentrations (25, 50, 75 and 100 µg/mL) of the test compounds and incubated for 150 min at 25◦C and 1mL of the reaction mixture was treated with 1 mL of Griess reagent (1%) sulfanilamide, 2% H3PO4 and 0.1% naphthylethylenediamine dihydrochloride). The absorbance of the chromophore was measured at 546 nm.
Hydrogen peroxide scavenging activity
The H2O2 scavenging ability of the test compounds was determined according to the literature method . A solution of H2O2 (40 mM) was prepared in phosphate buffer (pH 7.4). 25, 50, 75 and 100 µg/mL concentrations of the test compounds in 3.4 mL phosphate buffer were added to H2O2 solution (0.6 ml, 40 mM). The absorbance value of the reaction mixture was recorded at 230 nm.
RESULTS AND DISCUSSION
Ultra violet–visible spectroscopy of As-AgNPs
UV-visible spectroscopy is a straightforward technique for the detection and also for confirming the formation of nanoparticles. The bio-reduction of Ag+ ions by using the leaf extract of Andrographis serpyllifolia was monitored from time to time by sampling of the 1 µl aliquots and the optical absorbance was recorded on the Nanodrop 8000 UV-vis spectrophotometer in 220 – 700 nm wavelength range. The UV-visible spectra (Fig. 2) of the bioreduced As-AgNPs solution showed an absorbance peak at 421 nm which is a characteristic surface plasmon resonance (SPR) peak of silver nanoparticles, which confirms their biosynthesis. The size and shape of the As-AgNPs reflects on the absorbance peak.  It well known that the SPR range of silver nanoparticles is between 390nm to 470nm, earlier reports on biosynthesized AgNPs also reveals the similar results that if the SPR of AgNPs is in-between 400nm to 450 nm range, it reveals that the AgNPs are small and spherical in shape, in the size range of 20nm to 100nm . In the present study the SPR of As-AgNPs is 421nm, it indicates that the nanoparticles are small and spherical in shape this further confirmed by TEM analysis and Particle size analysis.
Fourier transform infra-red (FTIR) spectra ananlysis of As-AgNPs
The FTIR analysis was performed by using Brucker Tensor 27. The FTIR spectral analysis of As-AgNPs (Fig. 3) showed intensive peaks at 3844, 3735, 3292, 2921, 2351, 1640, 1528, 1408, 1242, 1030 and 534 cm-1 respectively. The sharp peaks at, 3844 cm-1, 3292 cm-1, 2921 cm-1, 2351 cm-1 can be assigned to hydroxyl group of alkanes such as O-H and C-H stretching of the –alkenes group, 1640 cm-1 can be assigned to C=C stretching of the alkenes group, 1520 cm-1, 1408 cm-1 can be assigned to Amide II stretching, 1242 cm-1 can be assigned to amide III stretching, 1030 cm-1 can be assigned to –C–O–C–stretching and –C=C– of the amide group.[15, 38] Therefore the FTIR analysis clearly revealed that the phytochemicals like amides, carboxylic acids, ether groups, hydroxyl groups and alcohol groups present in the leaf extract were mainly responsible for the reduction of silver ions (Ag+) into nanoscale silver particles (Ago). From the above results, it may be concluded that Andrographolides are the major bioactive compounds responsible for capping and efficient stabilization of As-AgNPs by acting as capping agents .
Particle size and Zeta potential analysis of As- AgNPs
The particle size of the biosynthesized As-AgNPs is detected by the intensity and laser diffraction method using the biosynthesized colloidal solution in which the As-AgNPs are poly-dispersed in mixture solution. The distribution of As-AgNPs are in the range of 1nm to 12 nm in size with and the average size of synthesized As-AgNPs was found to be 6.1 nm (Fig. 4) with and PI value of 0.437 (poly disperse index). Furthur the zeta potential analysis of As-AgNPs was detected to be -27.2 mV, due to its high negative zeta potential it prevent the As- AgNPs from agglomeration in the medium, leading to long term stability, because of the electrostatic repulsive force between the As-AgNPs. The zeta potential of As-AgNPs of Andrographis serpyllifolia leaf extract was found to be -27.2 mV (Fig. 5) which is similar to earlier reports [7-9, 16]
Energy dispersive X-Ray spectroscopy (EDX) of As-AgNPs
The EDX analysis were performed with the help of the Oxford Inca Penta FeTX3 EDS instrument attached to Carl Zeiss EVO MA 15 scanning electron microscope (200kV) with a line resolution of 2.32 (in Ao). The EDX data revealed that, very strong signal to silver and weak signals to other elements indicates the complete reduction of silver ions to elemental silver that is As-AgNPs (Fig. 6). The results are similar to earlier reports [10-13, 16]
X-Ray diffraction analysis (XRD) of As-AgNPs
XRD is an advanced spectroscopic technique used to evaluate the crystalline nature of biosynthesized As-AgNPs. The XRD pattern obtained revealed that As-AgNPs are face centered cubic structures in nature (Fig. 7) and that five characteristic diffraction peaks indexed to (111), (220), (311), (420), (422) and (220) planes of face centered cubic crystal lattice of Silver (JCPDS card No.01-1167).[13, 39-40].
Transmission electron microscopy (TEM) analysis of As-AgNPs
TEM analysis was performed to determine the shape and size of the biosynthesized As-AgNPs. TEM analysis was carried out using 200 kV FEI-Tecnai G@ 20 S-TWIN High resolution TEM (VIT, Vellore). The TEM image analysis also revealed that the As-AgNPs are roughly spherical in shape the As-AgNPs size is in-between 2 nm to 20 nm with an average size of 6 ± ٢ nm (Fig. 8), the results are similar to particle size analysis. The SAED pattern analysis also reveals that the particles have different planes of cubic crystal lattice which is already confirmed by XRD analysis.
Biomedical Applications of As-AgNPs synthesized from Andrographis serpyllifolia
The antimicrobial activity and the antioxidant activity were performed from the As-AgNPs biosynthesized from leaf extract of Andrographis serpyllifolia.
Antimicrobial activity of As-AgNPs
To reveal the biomedical importance of the As-AgNPS biosynthesized from Andrographis serpyllifolia, antimicrobial activity was performed against four bacterial species viz. Gram positive Staphylococcus aureus, Lactobacillus, Enterococcus and Gram negativeEscherichia coli, by the Kirby – Bauer disc diffusion method. The synthesized As-AgNPs from Andrographis serpyllifolia exhibited excellent antibacterial activity against Lactobacillus and Staphylococcus aureus and zone of inhibition (ZOI) of diameters 8 mm,12 mm,14 mm and 20 mm and 7mm, 12 mm, 14mm and 16mm respectively. The As-AgNPs showed a very good antibacterial activity against both Gram +ve and Gram –ve bacteria (Fig. 9). The As-AgNPs exhibited almost moderate activity with that of Amoxyclav (Table 1). The results were similar to the earlier reports [7-10, 16-17]
Antioxidant activity of biosynthesized As-AgNPs
In the present study we determined the free radical scavenging activity that is the measurement of the antioxidant levels in leaf extracts of Andrographis serpyllifolia and biosynthesized As-AgNPs, by different method like DPPH, H2O2 and NO antioxidant activity. Plants are usually rich in polyphenols. In the present study, the Andrographis serpyllifolia plant has a very high content of Andrographolides which are supposed to poses various medical and pharmaceutical functions. These important plant components give up hydrogen atoms from their hydroxyl groups to free radicals and form stable phenoxyl radicals. The results revealed that both plant leaf extract and biosynthesized As-AgNPs have excellent antioxidant activity. DPPH method proves to be the more efficient method when compared with H2O2 and NO antioxidant activity . The previous reports on plant mediated humic acids also have very good antioxidant activity . The antioxidant acivity was more with bioinspired As-AgNPs when compared with plant leaf extract. (Fig. 10) and (Table 2).
SUMMARY AND CONCLUSION
The biofabricated silver nanoparticles (AgNPs) using leaf extract of Andrographis serpyllifolia where characterized by using different spectroscopic methods. The biosynthesized green As-AgNPs were spherical in shape and the average size was depicted to be 6 ± 2 nm. The As-AgNPs are highly stable due the negative Zeta potential indicating that the As-AgNPs are polydispered in nature. Further the antimicrobial study of As-AgNPs on different bacterial strains indicates effective antimicrobial activity, As-AgNPs also showed excellent antioxidant activity by DPPH, Nitric oxide and Hydrogen peroxide method. Therefore the biosynthesized As-AgNPs can be valuable for a variety of biomedical applications.
The authors are grateful to DST PURSE centre, Sri Venkateswara University, Tirupati, for providing facility to carryout apart of research
CONFLICT OF INTEREST
All Authors don’t have any conflict of interest.