Green Synthesis and Antimicrobial Potential of Silver Nanoparticles with Boerhavia procumbens extract

© The Author(s) 2020. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Rizwan et al. | J Pure Appl Microbiol | 14(2):1437-1451 | June 2020 Article 6418 | https://doi.org/10.22207/JPAM.14.2.42


INTROdUCTION
Medicinal plants have been widely used and considered the most common sources of medicine in the pharmaceutical industry  (Bajguz, 2007;Cushnie et al., 2014). The composition of plant secondary metabolites depends on the species, association with microbes, and soil type (Zhao et al., 2011;Morsy, 2014). These bioactive compounds have potential against a wide range of bacterial infections and the best source for the formulation of drugs, declared by the World Health Organization (Iwu et al., 1999;WHO Traditional Medicine Strategy, 2002).
Nanotechnology or nanoscale technology is a strong field of novel research dealing with design, manipulation, and synthesis of particles with 1-100 nm (Sultana et al., 2019). Nanoparticles (NPs) have several uses in cosmetics, health care, food and feed, mechanics, optics, environmental health, drug-gene delivery, biomedical sciences, electronics, space industries, chemical industries, optoelectronics, energy science, catalysis, singleelectron transistors, nonlinear optical devices and photo-electrochemical applications (Iravani et al., 2014). Nanotechnology finds its importance by generating nano-products and NPs with novel and size-related physicochemical traits in contrast to bigger molecules (Chouhan, 2018).
T h e m o st i m p o r ta nt b ra n c h o f nanotechnology is plant-mediated green synthesis. Due to the environmentally friendly and practical cost, it has gained more importance (Chanel et al., 2017). Green synthesis has many advantages, i.e., less toxicity, cost-effective and straightforward, less energy consumption, perform activities under moderate conditions, and combined the effect of nanoparticles and plant bioactive constituents. Furthermore, green synthesized silver nanoparticles possess more biological potential than nanoparticles synthesized by chemical methods (Choudhury et al., 2016).
Silver NPs (AgNPs) are of interest due to their novel chemical, physical, and biological traits compared to their macro-counterparts (Sultana et al., 2019). AgNPs possess distinct physicochemical properties, comprising a high thermal and electrical conductivity, surface-chemical stability, enhanced Raman scattering, catalytic activity, and non-linear optical properties (Khattak et al., 2019). Those characteristics are responsible for their role in the formation of inks, in medical imaging and microelectronics. Besides, AgNPs reveal widespectrum fungicidal and bactericidal potential, making them highly accessible in functional products (Tran and Le, 2013).
Boerhavia procumbens belong to the family Nyctaginaceae, nearby termed Biskhpara/ Jangli, and spreading hogweed. Leaves are large, broad, and rounded beneath with a vigorous shape. Its branches (about 3 ft.) are glabrous and slender. The branches are long, glabrous, pubescent, stout, or viscous, with 1-2 inches leaves. They are oval, dull-witted, acute, and ordinarily cordate green beneath with panicle inflorescence (Fig. 1). Its habitat Africa, Pakistan, the USA, and India. In Pakistan, it could be found in Peshawar, Attock, Hazara, Sind, Baluchistan, Multan, Thal to Kurram, and Rawalpindi. Its color is purplish-red, and the blooming period is January to August (Hussain et al., 2012). Boerhavia procumbens has a lot of pharmaceutical and health-related benefits. Medicinal applications of Boerhavia procumbens include diaphoretic, diuretic, expectorant, emetic, laxative, stomachic, rejuvenates and purgative (Khalil et al., 2017;Wajid et al., 2017;Khan et al., 2020). It is mentioned to be effective against cardiac disorders, asthma, hemorrhoids, edema, eye diseases, insomnia, jaundice, kidney stone, nervous system related disorders, cough, against snake venom, urethritis, skin related diseases, intestinal, anemia, and colic kidney disorders The purpose of the current investigation was to carry out green synthesize of AgNPs using aqueous extract of Boerhavia procumbens and to study the antimicrobial potential of different fractions and synthesized AgNPs against pathogenic bacteria and fungal species.

Plant collection
B. procumbence was collected from the botanical garden of the Medicinal Botanic Centre of Pakistan Council for Scientific and Industrial Research (PCSIR), Peshawar, Pakistan. The plant was identified in the Department of Botany, University of Peshawar. The leaves having a voucher name BP554 was submitted for future correspondence. The freshly collected plant was washed carefully, then air-dried for 15 days under the shed (Pirtarighat et al., 2019). The dried plant was grounded to make a coarse powder using an electric grinding machine.

Preparation of plant extract
The crude extracts were prepared with different solvents, including methanol, water, ethanol, ethyl acetate, chloroform, and n-hexane. All solvents were taken into flasks, and plant material was soaked in flasks for three days. The solvents were decanted, and the procedure was repeated four times for each solvent. All extracts were concentrated using a rotary evaporator at 45°C under vacuum. The extracts were kept in the refrigerator for further analysis (Pirtarighat et al., 2019).

Moisture determination
Dried powdered B. procumbens (5 g) was pre-weighed in the Petri dish and kept at 105-110˚C for three h. Moisture (%) was calculated regarding the air-dried sample ( determination of extractive value The total weight of 5 g of each dried powder of plant material was extracted for about 24 h with 100 mL of respective solvent (methanol, ethanol, ethyl acetate, chloroform, and n-hexane). The flasks were shaken, and the mixture allowed to stand overnight, followed by recovery of solvents by decantation and filtration. The filtrates were dried, and the extract was weighted in percentage (Singh et al., 2013). % Extract = X 100 Wight of the extract (g) Wight of plant material (g)

determination of ash
The total weight of 5 g of dried powder of B. procumbens was burnt at 600˚C in a furnace for three h followed by cooling. The ash was weighed, and the percentage calculated regarding the air-

Preparation of silver nanoparticles (AgNO 3 )
Ten g of the plant powder was boiled with 100 mL deionized distilled water. The resultant aqueous solution was filtered using Whatman filter paper three times to remove all the insoluble material. This extract was added to a 10 -3 M silver nitrate (AgNO 3 ) solution. Analytical grade AgNO3 (Molecular weight 169.87 g/mol) was purchased from Merck (Darmstadt, Germany). The solution was heated for three h in a water bath at 60°C. The solution color changed from light yellow to brown-reddish. It showed the reduction of pure Ag (I) ions. The reduced ions were monitored and measured using UV-visible spectra of the mixture of plant extract and AgNO 3 solution at regular intervals. Peaks were measured on UV-Vis spectra from 200 nm and 700 nm (Shakeel et al., 2015).

Evaluation of antimicrobial activity
Antimicrobial activities were performed using well diffusion method for both B. procumbens crude extracts and AgNPs. The ethyl acetate, methanol, ethanol, water, chloroform, and n-hexane extracts of the plant and AgNPs solutions of B. procumbens were applied against ten pathogenic bacteria (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Citrobacter braakii, Proteus vulgaris, Salmonella Typhi, Salmonella Para typhi, Vibrio cholerae, and Providencia stuartii) and five fungal strains (Candida albicans, Alternaria alternata, Aspergillus flavus, Aspergillus niger, and Aspergillus oryzae). The agar media was prepared then autoclaved and dispensed into sterilized plates in Laminar flow hood (LFH) and left for the solidification. The test microorganism uniform lawn was arranged on the surface of nutrient agar media in Petri plates with the help of a sterile swab. Uniform wells of 6 mm were bored in each plate, with sterile metallic borer. Ten mg of test samples (fraction and AgNPs) were prepared in 1.0 mL of DMSO (10%) and added into particular wells. Petri dishes were incubated for 16 h at 37°C. The zones were determined after the incubation period from the width of the wells. The formula documented the percent inhibition: Zone of inhibition of standard

RESULTS
The current study was performed to evaluate the antimicrobial potential of B. purocumbens crude extract, to synthesize AgNPs from B. purocumbens extract and to determine their biological effects against pathogenic bacteria and fungi. Leave extracts were prepared in methanol, ethyl acetate, ethanol, chloroform, n-hexane, and water. Ash (19.38%), moisture (8.2%), and extractive values were also determined ( Table 1). The FTIR of all extracts was performed    using FTIR Model IR Pretige-21 (Shimadzu, Japan). Different peaks of functional groups were detected in each fraction (Fig. 2-6).

Phytochemical analysis of B. purocumbens extract
The results of the phytochemical analyses, shown in Table 2, reveal that tannins were present in methanol, ethanol, ethyl acetate, chloroform extracts. Flavonoids were absent in the n-hexane extract, while present in all other extracts. Alkaloids and terpenoids were absent in all extracts. Saponins were absent in the n-hexane extract, while present in methanol, ethanol, ethyl acetate, chloroform, and water extracts. Steroids were present is less quantity in n-hexane extract, while strongly found in all the remaining extracts. Quinones was absent in n-hexane extract, while detected in chloroform, ethyl acetate, ethanol, methanol, and aqueous extracts. Phenols were present in much higher concentration in methanol and ethanol extracts, while moderately present in n-hexane and EtOAc extracts, and least detected in chloroform and water extracts.

FTIR analysis of B. purocumbens extract
All fraction of crude extract of B. purocumbens were evaluated for the presence of functional groups using FTIR (Fig 2-7). The Fig. of each fraction was analyzed from its IR values and presented in a tabulated form (Table 5-10).

FTIR of AgNPs
The FTIR analysis was carried out to identify AgNPs associated biomolecules. The  results showed that different compounds were detected in the AgNPs sample, i.e., phenols, alcohols, carboxylic acid, ketones, alkenes, nitro group, and ethers. After comparing the spectra of AgNPs and aqueous extract, it has been confirmed that AgNPs are synthesized. The differences of IR spectra of AgNPs and aqueous extract were due to the compounds of aqueous extracts that have been oxidized by silver ions. Some of the peaks present in aqueous IR spectra were absent in AgNPs extracts. This means that AgNPs have reacted with these groups (Fig. 7-8). The FTIR analysis of AgNPS was interpreted using IR values and presented in Table 11. pH and color Synthesized AgNPs can also be confirmed physically by pH and color changes. AgNPs change their color from yellowish to dark brown, and pH changes from 6 to 5.5. The change in pH and color confirms the presence of AgNPs.

UV-Vis Spectroscopy of AgNPs
The UV-Vis spectroscopy is performed to characterize the AgNPs by the surface plasmon resonance (SPR) with a spectrum of 200 to 700 nm. The maximum peak absorbance with the high band was noted at 395 nm (Fig. 9).

Scanning Electron Microscopy (SEM) of AgNPs
The SEM picture shows the shape and size of the AgNPs. The SEM images showed spherical shape AgNPs in scattered form focused on 0.5 µm of area, with diameter ranges from 20-80 nm (Fig.  10).

Antimicrobial activity
For antimicrobial activities, B. procumbens extracts obtained from different solvents, as well as synthesized AgNPs were screened against bacterial and fungal pathogens (Table 3 and 4). B. Percumbens plant extracts prepared using different solvents, including water, MeOH, CHCl 3 , EtOH, EtOAc, and n-hexane. MeOH and EtOH fractions resulted in significant activity against pathogenic bacteria and fungi. The extracts of the remaining fraction have no or very few activities. Furthermore, it was also clear that B. Percumbens synthesized AgNPs were more active than crude extracts (Tables  3 and 4). B. Percumbens synthesized AgNPs can be used to enhance the antimicrobial potential of plant constituents against a wide range of infections.

dISCUSSION
Medicinal plants have attracted significant attention to science communities due to the promising potential in life science (Antony and Sing., 2011). Pakistan is very rich in natural wealth because of it is excellent geographical condition, multiple ecological niches, and soil conditions. It     , 2007). Nanotechnology continues to allow researchers and technologists to find new materials and essential benefits to people. However, it is also an essential part to consider the potential harmful impacts of nanoscale generated products on health as well as our environment (Hull and Bowman, 2018). Due to these facts, it is essential to determine how to decrease dangerous impacts interred by new materials. In the present study, green synthesis of AgNPs was adopted to reduce the negative impact on health and environment because this is the safest method of preparation of AgNPs. The procedure used by Ahmed et al. (2016) for the synthesis of AgNPs by the chemical method was followed. The methodology for the synthesis of AgNPs was the same, but instead of chemical synthesis, the green synthesis method was used in the present study. The most valuable NPs for medical uses are AgNPs, which are well known for their high antimicrobial activity. AgNPs has a remarkable option for the development of bioactive and novel drugs due to their broad-spectrum activities. Silver ion is famous as a metal ion that shows antimicrobial, antifungal, and anti-algal properties. Mainly, it has been widely used as silver nitrate solution in water, which has sterilizing and also disinfecting activities (Kheybari, 2010). Hussain et al. (2012) revealed that roots containing oil of B. procumbens are composed of methyl ester derivatives of fatty acids. Eighteen types of different components were found and quantified as well. The most important and abundant was the methyl ester of linoleic acid (9%). In the present study, the phytochemical analysis revealed different types of phytochemicals, in which steroids are of great interest nowadays. B. procumbens was used as an entire plant for the process of phytochemical analysis, the plant contains eight important phytochemicals and having good antimicrobial activity. Furthermore, this ability was enhanced by synthesizing the  Plants having different medicallyimported bioactive components could be used to treat severe human pathogenic diseases. There are two types of categories of phytochemicals: i.e., the primary and secondary compounds. The primary compounds contain proteins, chlorophyll, sugar, and amino acids. Secondary compounds include alkaloids and terpenoids (Wadood et al., 2013). The objective of the present study was the same as both studies investigated the phytochemical   profile. Important phytochemicals were identified, among them are terpenoids, flavonoids, reducing sugars, and alkaloids. These compounds serve as a plant defense against microorganisms, insects, and herbivores. For example, phenolics are the simplest phytochemicals, which are effective against viruses (Wild, 1994), bacteria (Thomson, 1978), and fungi (Duke, 1985). Catechol and pyrogallol are both hydroxylated phenols, toxic to microorganisms. The mechanism of action is enzyme inhibition in different ways. Flavonoids are effective antimicrobial due to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell walls (Tsuchiya et al. 1996). Furthermore, quinones cause inactivation of the (Kazmi et al. 1994), tannins could inactivate microbial adhesins, enzymes, cell envelope transport proteins (Nonaka et al., 1990) and alkaloids intercalate with DNA (Phillipson and O'Neil, 1987).
The clinical trial concerning the antimicrobial significance of AgNPs was carried out with the zone of inhibition produced by pathogenic organisms (gram-negative and grampositive bacteria) and some disease-causing fungus. (Geoprincy, 2013). The present study aimed to evaluate the antimicrobial activity of B. procumbens. Among extracts used, methanol and ethanol showed significant activity against targeted pathogenic bacteria and fungi. The extract of the remaining fractions did not show or showed less activity. Interestingly, the prepared AgNPs from B. procumbens showed better results as compared to crude extracts.