Nanotechnology and nanoscience are due to their numerous uses in medicine, engineering, and water pollution sensors and their expanding research fields. In this study, the essential oil, methanolic extract, and biosynthesized silver/silver oxide nanostructures (Ag/AgO NSs) using the aqueous extract of the plant were prepared. The phytochemical compounds of the extract and essential oil were analyzed using gas chromatography/mass spectrometry (GC/MS), respectively. The GC/MS technique identified 34 compounds in the essential oil of the plant with the major constituents including oleic acid (18.5%), palmitic acid (11.08%), phytone (6.64%), p-Vinylguaiacol (6.4%), and phytol (4.23%). After the phytochemical identification, the total flavonoid and polyphenol content of the extract was determined, too. Prodelphinidin B3 compound in the M. sylvestris extract was analyzed and detected by high-performance liquid chromatography/ultraviolet detector (HPLC-UV), at a retention time of around 10 minutes. In addition, M. sylvestris extract was used for green synthesis of Ag/AgO NSs. The as-prepared NPs were characterized using X-ray Diffraction (XRD), UV-visible absorption spectroscopy, scanning electron microscope equipped with energy-dispersive X-ray spectroscopy (SEM/EDS), Brunauer Emmett Teller (BET), Barrett-Joyner-Halenda (BJH) and Fourier transform infrared (FT-IR) analyses. Surface plasmon resonance (SPR) absorption at λmax 320 nm in the UV-vis spectra confirm the formation of Ag/AgO NSs. The crystalline structure of Ag/AgO NSs was confirmed by XRD analysis. The small size, with three different Williamson-Hall, Sherrer and SEM/EDS methods, size of nanoparticles were obtained 64.16, 44.33 and 50 nm respectively. Besides, that spherical shape of Ag NPs with good size distribution was observed in the SEM/EDS analysis. The small size, around 50 nm, and spherical shape of Ag/AgO NSs with good size distribution was observed in the SEM/EDS analysis. Besides, the antibacterial activity of the extract was evaluated against three pathogenic bacteria, by disk diffusion method. Significant antibacterial activity was observed for the prepared extracts of M. sylvestris against the bacteria (Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa), and the results were compared with the known antibiotics such as amoxicillin, cephalexin, erythromycin, and fluconazole.
Research Article
Green synthesis of silver/silver oxide nanostructures using the Malva sylvestris extract prior to simultaneous distillation extraction: synthesis, phytochemical and biological analysis
https://doi.org/10.21203/rs.3.rs-4861630/v1
This work is licensed under a CC BY 4.0 License
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Malva sylvestris
Green synthesis
silver nanoparticles
Prodelphinidin B3
Antimicrobial activity
Essential oil
- Green synthesis of Ag/AgO nanostructures were carried out using the aqueous extract of Malva sylvestris.
- Phytochemical compositions and antimicrobial potential of Malva sylvestris were evaluated.
- The prodelphinidin B3 compound in the M. sylvestris extract was detected by the HPLC.
- GC/MS analysis identified 34 major constituents in the essential oil of the plant.
Malva sylvestris (Mallow) (Scheme 1) belongs to the genus Malva, which contains 100 species. The well-acknowledged genus Malvaceae belongs to the family Malva and is geographically distributed in South-west Asia, North Africa, and Europe (Rodrigues et al 2003). Herbs can be used in the form of plant extracts or as their active components. Most of the world’s population uses herbal materials due to their strong antimicrobial properties and primary healthcare benefits (Parham et al 2020). The extract of this plant has high phenolic content and high antimicrobial properties. The antibacterial effect of this herb has been reported against a variety of bacteria (Gram-positive and Gram-negative) including S. aureus, E. coli, Bacillus cereus, Salmonella typhimurium, Klebsiella pneumoniae, Listeria monocytogenes, Proteus vulgaris, Streptococcus pyogenes, Micrococcus luteus, Mycobacterium smegmatis, and Pseudomonas aeruginosa (Misak et al 2011, Walter et al 2011, Paul et al 2016, ). The antioxidant and antimicrobial activities of M. sylvestris are attributed to its chemical components including ß-carotene, vitamin E, flavonoids, polyphenols, and vitamin C (Nasiri et al 2015). It has traditionally been applied to treat a variety of ailments such as toothache, laxative, cough, cut wound healing, burns, and colds (Cecotti et al 2016).
The common M. sylvestris is known for its various medicinal properties, including choleretic, laxative, diuretic, and spasmolytic effects. It is also used as an expectorant, antitussive, bronchodilator, and anti-diarrheal, and is recommended for skin care and acne treatment due to its antiseptic, demulcent, and emollient properties (Quave et al 2008, Leporatti et al 2009). Studies have also looked into the potential effects of M. sylvestris crude extract fractions on COVID-19, as well as its anti-HIV bioassay using epithelial (Irfan et al 2021).
Phytochemical studies have revealed that this herb contains polysaccharides, anthocyanins, coumarins, anthocyanidins, arabinogalactans mucilage, tannins, leucoanthocyanidines, flavones, mucilage, flavonols, and terpenoids such as monoterpenes, sesquiterpenes, and diterpenes (Gasparetto et al 2012, Prudenteet al 2013). Condensed tannins or proanthocyanidins are important polyphenolic compounds found in plant-derived foods like beverages, fruits, and cereals (Siupp 1977). Prodelphinidins, which belong to the condensed tannins or proanthocyanidin sub-group, have been isolated from various plants and are known for their antioxidant activity (Teixeira et al 2016).
The following plant compounds have been isolated and reported in studies: prodelphinidin B1 from Cistus incanus, stryphnodendron adstringens, Lotus pedunculatus; prodelphinidin B2 from S. adstringens, L. pedunculatus; prodelphinidin B3 from Quercus dentata and Sorghum vulgare seeds; and prodelphinidin B4 from Vicia faba, Ribes nigrum, and S. adstringens (Makabe et al 2016). The isolated prodelphinidin fraction exhibits various biological activities, with antioxidant activity being considered one of the most promising (Savickiene et al 2024), especially in its efficacy against Porphyromonas gingivalis and Streptococcus salivarius.
Essential oils are important compounds found in medicinal plants, possessing various biological activities (Thériault et al 2006). These compounds are hydrocarbons composed of carbon, hydrogen, and oxygen, belonging to the terpenoid group, including monoterpenoids and sesquiterpenoids (Walter 2015). These volatile substances have antioxidant activity and protect cells from the harmful effects of oxygen-free radicals (Eslami et al 2010).
In recent years, there has been a growing interest in green synthesis by various research groups due to its advantages. Plants contain a wide range of secondary metabolites such as phenolics, polysaccharides, and flavonoids with redox capacity (Machado et al 2013, Karami et al 2020, Amirhosseini et al 2023, Madhavi et al 2013). Green synthesis involves the use of reducing agents, natural stabilizers, and capping agents extracted from the plants (Kuang et al 2014, Oroumi et al 2024). The present natural products and/or secondary metabolites can be used instead of some chemical precursors. In comparison to chemical and physical procedures, green synthesis is cost-effective, environmentally friendly, and does not require high temperatures and pressure. This procedure is free from toxic chemicals. Furthermore, green-synthesized nanoparticles are inexpensive and resourceful. So far, the biological synthesis of silver nanoparticles (Ag NPs) has been carried out using the plants such as Achras sapota L. (Thakore et al 2019), Piper longum L. (Jacob et al 2012), and many other nanoparticals with biocidal properties such as, copper oxide, Fe3O4, and silver/carbon nanoparticles (Benhammada et al 2022, Mousavi et al 2020, Moaddab et al 2021), and many other species plant with biological properties such as Malva parviflora, Malva verticillata (Helmy et al 2021, Sk et al 2020).
In the current study, the methanolic extract of the M. sylvestris plant as well as its essential oil were prepared. The essential oil was obtained using the simultaneous distillation extraction (SDE) technique. Then, the phytochemical compounds were analyzed and identified using high-performance liquid chromatography combined with detector-coupled Ultraviolet (HPLC-UV), and gas chromatography combined with mass spectrometry (GC/MS). The qualitative and quantitative analysis of the prodelphinidin in M. sylvestris was conducted, too. Additionally, an extract plant-mediated approach was used to synthesize green silver/silver oxide nanostructures (Ag/AgO NSs). The as-synthesized NSs were characterized using various techniques.
2. 1. Plant material
M. sylvestris is an herbaceous and annual or perennial plant species (Fig. 1). It is 40–60 cm in height, characterized by long laxly stems, circular and jagged leaves spaced along young stems, and blue or purple flowers. It represents all kinds of Malvaceae grown all over Iran originally (Surmaghi et al 2011). The aerial parts of M. sylvestris were harvested in April 2021, during the flowering period from the Basht region (N 37′′ 21′ 30° / 42′′ 9′ 51° W) Fig. S1.
(Fig. 1)
2. 2. Essential oil extraction
The essential oil extraction process was carried out eight times, with each batch consisting of 200 g of plant material. The process involved drying the aerial parts of the plant at room temperature (25°C) and then performing hydrodistillation using an SDE apparatus for at least 4 hours. The obtained essential oils were then dried over anhydrous magnesium sulfate and stored in a black bottle at 4°C for further use. The qualitative and quantitative analyses of the essential oil composition profile were conducted using the GC/Mass spectrometry (GC/MS) method (Fadli et al 2012). The analysis was conducted using GC/MS under appropriate conditions (carrier gas, injector, times, and temperatures). The identification of essential oil compounds carried out through a mass spectrum comparison with the National Institute of Standards and Technology (NIST) and Wiley Library data (Adams et al 2007).
2.3. Extract preparation
The leaf extract of M. sylvestris was obtained using the Soxhlet apparatus and maceration.
i. Preparation of the plant extract
The plant extract was prepared for UV-Vis, FT-IR analysis, HPLC, and antibacterial testing using the dynamic maceration method.
ii. Preparation of the extraction for HPLC analysis
For the HPLC analysis, 25g of finely powdered pericarp of the plant was extracted through dynamic maceration and stored in the refrigerator for testing.
iii. Preparation of the Aqueous Extraction for Ag/AgO NSs synthesis
The aqueous extraction of the M. sylvestris plant powder was performed using the ultrasonic method. A ratio of 10g: 450mL between M. sylvestris plant and deionized water was used. The aqueous extract was sonicated for 15 minutes (3 run), at a temperature of approximately 40°C. After extraction, the mixture was filtered with Whatman filter papers degree 42, which was then used to synthesize Ag/AgO NSs.
iv. Preparation of the antibacterial tests
For the antibacterial tests, Mueller Hinton agar (MHA), Mueller Hinton broth (MHB), gallic acid, quercetin, and prodelphinidin B3 (PDB3) were sourced from Sigma Aldrich (Saint Louis), and all organic solvents were procured from Merck Millipore (Darmstadt, Germany).
2.4. Total phenol and flavonoid content
The total phenol content was determined using the Folin–Ciocalteu method as outlined by Rajabi in 2019e. A 500 µL sample of the leaf extract was transferred into a test tube, followed by the addition of 1.0 mL of Folin–Ciocalteu reagent (diluted at a ratio of 1:10 with deionized water). The contents were mixed and left at room temperature for 5 minutes. Then, 1.0 mL of 6% (w/v) sodium carbonate was added to the mixture, gently shaken, and left at room temperature for 90 minutes. The absorbance was measured at 200 to 800 nm using a UV–Vis spectrophotometer (JASCO-730, Japan). Gallic acid was utilized as the standard reference, and the total phenol content was expressed as mg gallic acid equivalent (FAE)/g.
The total flavonoid content in the extract was determined using the aluminum chloride colorimetric method described by Loizzo et al in 2016 and Rajabi et al in 2021. For this, 1.0 mL of leaf extract was transferred to a test tube, followed by the addition of 2.0 mL of methanol, 1.0 mL of 10% aluminum chloride, 1.0 mL of 1.0 M sodium acetate, and 2.0 mL of distilled water. After 30 minutes at room temperature, the absorbance of the reaction mixture was measured using a UV–Vis spectrophotometer. Quercetin was used as the standard compound, and the total flavonoid content was expressed as 0.005g quercetin equivalent (RE)/g.
2.5. Biosynthesis of Ag/AgO NSs
In an Erlenmeyer flask, 90mL of 1mM silver nitrate (AgNO3) solution was mixed with 10mL of M. sylvestris extract. The synthesis took place in an alkaline media with 25% ammonia (NH3) to maintain a pH of 9. The solution was stirred at 500 rpm using a magnetic stirrer at a temperature of approximately 80°C. The obvious change in the plant extract from light yellow to brown may be due to the reduction of Ag+ ions to Ag◦ (Ag NPs). The solution containing the prepared nanoparticles was then centrifuged at 10000 rpm for 15 minutes, and the supernatant was discarded.
The formed M. sylvestris/ Ag NPs was analyzed using the FT-IR analysis, by a PerkinElmer Spectrum 2 FTIR spectrophotometer with the Attenuated Total Reflectance (ATR) in the wavenumber range of 400–4000 cm− 1. The Rigaku Ultima IV, Japan X-ray multipurpose θ-θ X-ray diffractometer was used to analyze the X-ray diffraction pattern from 15 to 85. UV–Vis spectra were analyzed by the UV-visible (Jasco 730, Japan) spectrophotometer in the range of 200–800 nm. The particle size, morphology, and lattice spacing were analyzed using a scanning electron microscope (ISP, M-300, Korea) equipped with energy-dispersive X-ray spectroscopy (SEM/EDS), under an accelerating voltage of 30 kV.
2.6. Antibacterial inhibitory effects
The antibacterial activities of plant extracts against three pathogenic bacteria (one Gram-positive and two Gram-negative) were investigated using the agar disk diffusion method (Rajabi et al 2019). The antibacterial activity testing was carried out using the agar-cup method. Each purified extract was dissolved in physiological saline. To determine the inhibition zone, pure Gram-negative and Gram-positive strains were taken as standard antibiotics for comparison of the results. All the extracts were screened for their antibacterial activities against Pseudomonas aeruginosa, Escherichia coli, and Bacillus subtilis. Sets of three dilutions (3.75, 7.5, 15 mg/mL) of M. sylvestris extract standard drugs were prepared in double distilled water using nutrient agar tubes. Mueller-Hinton sterile agar plates were seeded with indicator bacterial strains and left at 37°C for 3 hours. Control experiments were carried out under similar conditions using amoxicillin (30 µg), erythromycin (15 µg), cephalexin (30 µg), and fluconazole (25 µg) as standard drugs for antibacterial activity. The zones of growth inhibition around the disks were measured after incubation at 37°C for 24 hours. The sensitivities of the microorganism species to the plant extracts were determined by measuring the sizes of inhibitory zones on the agar surface around the disks, and values less than 6 mm were considered as not active against microorganisms.
2.7. FT-IR analysis
The dried powder (methanolic extract) of the test plant was used for FT-IR analysis. Additionally, 1.0 mg of the dried powder was encapsulated to prepare translucent sample discs. The powdered sample of the pellet was loaded into the FT-IR spectrometer (Perkin Elmer Spectrum 2, USA), with a scan span from 400 to 4000 cm− 1.
2.8. High-Performance Liquid Chromatography (HPLC) analysis
An isocratic HPLC profiling was carried out for the methanolic leaf extract of M. sylvestris at a flow rate of 0.8 mL/min. 20 mg of each substance was dissolved in a mobile phase containing a mixture of water and methanol (90:10, to 100 methanol v/v), and the injection volume was 20 µL. The C18 (4.6×250 mm, 5µm) column was kept at room temperature, and the eluent was detected at 280 nm with a run time of 10 minutes. The UV spectra of the extracted sample peaks were compared with the standard peak of PDB3.
3.1. UV- Vis absorption and FT-IR analysis of the extract
Spectroscopic techniques such as UV-Vis absorption and FT-IR spectroscopy can be used to identify the chemical compounds in plant extracts (Mohani et al 2014, Kumar et al 2020). FT-IR is a high-resolution analytical technique used to identify chemical constituents and elucidate structural compounds (Mamta et al 2012). These techniques offer rapid and nondestructive investigation to fingerprint plant extracts. The absorption and FT-IR spectra of the methanolic extract of M. sylvestris are illustrated in Fig. 2.
A broad and intense absorption band is visible around 300–400 nm, along with a sharp peak at 670 nm. These results can be used to characterize some compounds such as flavonoids and their derivatives in the methanolic extract. However, it's important to supplement the UV-Vis results with other techniques like GC/MS for proper characterization and constituent identification of the prepared extract (Mohani et al 2014). Similar observations have been reported for other plants such as Acorus calamus (Kumar et al 2020) and Micrococca mercurialis, Dillenia pentagyna (Mamta et al 2012).
The FT-IR peak values and corresponding functional groups are listed in Table S1. The FT-IR shows a broad peak at 3756.65 cm− 1, indicating the presence of free O-H stretching, and another peak at 3370.96 cm− 1 representing strong H-bonding. Additionally, strong peaks at 2858 and 2927 cm− 1 indicate the presence of C-H stretching. The observed peaks at 1334, 1395, and 1620 cm− 1 can be related to the presence of aromatic C = C stretch. Furthermore, sharp peaks at 998 and 1056.8 cm− 1 are assigned to the presence of = C-H vinyl, while the peak at 829 cm− 1 indicates the presence of C-C stretching. Based on the observed functional groups in the FT-IR spectrum, it can be confirmed that there are various secondary metabolites such as phenols, alkanes, alcohols, alkynes, and aromatic compounds in the methanol extract.
(Fig. 2)
3.2. UV-vis absorption and FT-IR analysis of the Ag/AgO NSs
The UV-visible spectroscopy results of the M. sylvestris aqueous extract before and after the synthesis of Ag/AgO NSs are presented in Fig. 3B. In the figure, it is evident that no significant absorption peak is observed in the plant extract, while Ag/AgO NSs shows an absorption peak at λmax at 320 nm Figre 3B. The presence of the maximum absorption peak in the curve obtained from the extract with Ag NPs at λmax of 320 nm is one of the simplest methods to demonstrate the synthesis of Ag NPs. The shifting changes or wavelength in peak intensity explain the types of functional groups involved in the binding mechanisms.
The FT-IR spectrum for M. sylestries extract exhibited absorption bands at 3005, 3679, 1576, 1388, 1080, 615, and 532 cm− 1, as illustrated in Fig. 3A. According to Yallappa et al 2015, the peaks ranging from 3365 cm− 1 to 2915 cm− 1 are related to the OH (hydroxyl) in the J. Sambac leaves extract. The peak at 1080 cm− 1 can be assigned to CO bending from the phenols and flavonoid compounds in M. sylestries extract. The strong, broad peaks observed at 1576 and 1388 cm− 1 correspond to the aromatic ring (C = C) of the phenols and flavonoids structure. The peak at 615 cm− 1 corresponds to the CH bending of aromatic bending outside the plane groups from the structure in M. sylestries extract.
The peak at 1385.37 cm− 1 corresponds to the C = C stretching (Khosravi et al 2023). The M. sylestries@Ag/AgO NSs synthesized showed stronger absorption intensity, and the FT-IR spectrum showed some shifting of the highest peaks (Fig. 3A). The peak shifted, indicating that the responsible functional groups were involved in the binding mechanism on the Ag/AgO NSs. After the synthesis process, the peaks at 3005, 3679, 1576, 1388, 1080, 615, and 532 cm− 1 shifted to 3000 to 3600, 1575, 1375, 1116, and 615 cm− 1 corresponding to C-O and O-H stretching, C-H stretching, the aromatic ring of the phenol and flavonoids structure, respectively (Shameli et al 2012). The peaks increased in the case of Ag/AgO NSs, suggesting that functional groups might also be bonded with the Ag/AgO NSs. Additionally, the FT-IR spectrum of the extract before and after the synthesis of Ag/AgO NSs is similar; however, the spectrum of the extract showed less intensity, indicating the activity of different types of phenolic and flavonoid compounds present in the extract (Slathia et al 2021, Atri et al 2023).
(Fig. 3)
3.3. XRD, EDS and BET elemental analysis
The XRD pattern reveals the determination of physicochemical properties of the Ag/AgO NSs by M. sylvestris plant extract. The XRD pattern of silver nanoparticles synthesized with M. sylvestris plant extract are depicted in Fig. 4, showing peaks (111), (200), (211), (220), (221), (013), and (311). The peaks at 2θ = 38.07°, 44.26°, 64.43°, and 77.16° are related to the face-centered cubic (fcc) structure of Ag NPs, which is in complete agreement with the standard X-ray diffraction pattern of silver (JCPDS, File No. 89-3722) (Alharthi et al 2020). The peaks at 2θ = 32.5°, 36°, 46°, 55°, 57°, 64° and 77.16° are related to the face-centered cubic (fcc) structure of AgO NPs, which is in complete agreement with the standard X-ray diffraction pattern of silver oxide (Abdalameer et al 2021).
The parameters of the unit cell are based on the information in the standard card No. 96-150-9147 Fig. 4 (B) (Alharthi et al 2020, Williams). The average crystallite size of the Ag NPs is calculated using Debye Scherrer’s formula.
$$\:\text{D}=\frac{\text{k}{\lambda\:}}{{\beta\:}\text{cos}{\theta\:}}$$
1
In the equation, D represents the grain size, where k = 0.9 is the crystal shape factor, λ is the wavelength of 1.5406 Å, β is the full width of the X-ray and is equivalent to the half maximum of the diffraction peak, and θ is the angle corresponding to the diffraction peak. The results of particle size calculation obtained from Scherrer's equation are 44.33 nm.
Williamson-Hall method: One of the drawbacks of Scherrer's method was that it related peak broadening only to grain size, while studies showed that peak width is related to network strains in addition to grain size. Williamson and Hall introduced the grain size and intra-lattice strains as the reason for the broadening of the peaks resulting from X-ray diffraction. Based on the presentation theory by Williamson-Hall, the width of the peak at half maximum intensity is a function of the particle size and also the intra-lattice strains.
$$\:\beta\:=\beta\:s+\beta\:d$$
2
\(\:\beta\:s\) And \(\:\beta\:d\) respectively, the broadening of the peak is due to the grain size and network strains. Stokes and Wilson defined the upper limit of network strain as follows:
$$\:{\epsilon\:}=\frac{\varDelta\:d}{d}=\frac{\beta\:s}{4{tan}\theta\:}$$
3
\(\:{\epsilon\:}=\frac{\varDelta\:\text{d}}{\text{d}}\) The relative change of the distance between the crystal plates is the lattice strain. With paying attention to relations 1 and 3, the size of grains \(\:\frac{1}{\text{cos}\theta\:}\) and strain with \(\:\frac{1}{\text{tan}\theta\:}\) which θ, Bragg's angle is proportional. By putting relations 1 and 3 in 2, we have:
$$\:\beta\:=\frac{k\lambda\:}{D}\text{cos}\theta\:+\:4\epsilon\:\text{tan}\theta\:$$
4
$$\:\beta\:\text{cos}\theta\:=\:\frac{k\lambda\:}{D}+4\epsilon\:\:\text{sin}\theta\:$$
5
Equation 5 is obtained by multiplying the sides of Eq. 4 in \(\:\text{cos}\theta\:\) for layer nanoparticles (\(\:\beta\:\text{cos}\theta\:-\beta\:\text{sin}\theta\:)\) Williamson. The graph of unbaked AgNps is shown in Fig. 5. By fitting the line to the graph data, the particle size and mesh strain can be calculated from the value of the width from the origin and D and the slope of the fitted line, respectively. Calculations of the particle size of the samples from the fitting line of Fig. 5 are given 64.16 nm.
The composition of the Ag/AgO NSs was analyzed using scanning SEM-EDX mapping as shown in Fig. 6A to investigate the element distribution of the NPs. The colored elemental mapping images in Fig. 6A show that Ag atoms (green) were evenly distributed throughout the entire NP structure, indicating that the Ag structure was alloyed in nature. Additionally, EDX elemental line scanning on a single particle Fig. 6B confirmed that the NP was composed of alloyed metal elements. The EDX data also indicated that the mole fraction of Ag was 2.31.
The presence of the Ag element in the mapping analysis demonstrated that the Ag NPs were synthesized, successfully. However, other elements such as C, O, K, Al, and N may be related to the extract of M. sylestries plant (Khosravi et al 2023).
Figure 7 (A) indicates the nitrogen adsorption–desorption isotherms of Ag NPs, which suggests the presence of mesoporous structures. Based on the BET analysis, surface area, the total pore volume, and mean pore diameter of the AgNPs were obtained at, 0.005 cm3 g− 1 0.738 m2 g− 1, and 27.04 nm, respectively. Besides, the Barrett–Joyner–Halenda (BJH) method is employed to study the pore size of a wide range of biogenic NPs, the pore size of Ag NPs produced by M. sylestries leaf extract was found to be 1.8 nm. Figure 7 (B)
(Fig. 4), (Fig. 5), (Fig. 6) and (Fig. 7)
3.4. Chemical composition of the essential oil
The essential oil extracted from the dried aerial parts of M. sylvestris was a light yellow oil obtained using SDE apparatus. The relative abundance and chemical composition of the essential oil are presented in Table 1 and Fig. 8. The GC/MS analysis identified 34 components, constituting 93.34% of the total oil composition. The main compounds in M. sylestris essential oil were oleic acid (18.5%), palmitic acid (11.08%), phytone (6.64%), p-pinylguaiacol (6.4%), and phytol (4.23%) as illustrated in Table 1. Additionally, the essential oil of the herbal M. sylestris contains phenolic and hydrocarbon compounds such as fatty acids. The results show that the essential oil of the flower, leaf, and fruits of M. sylestris contains main compounds including palmitic acid (18.8%), 4-vinyl guaiacol (14.0%), and α-linolenic acid (35.5%) (Table 2). These results are consistent with previous studies on M. sylestris essential oil composition obtained by the clevenger technique (Delfine et al 2017). They showed that the flowers had linoleic acid and palmitic acid, while the leaves were rich in α-linoleic acid. The chemical composition of the essential oil of the flowers of M. sylvestris collected in Japan has also been analyzed (Hasimi et al 2017). The findings show high percentages of hexadecanoic acid (10.5%) and hydrocarbons (25.4%) in the obtained essential oil.
The analysis of M. sylvestris shows that the essential oil composition in the Malva genus differs in terms of chemical profile compared to other species. M. sherardiana (Jain et al 2016) and Malva neglecta essential oils (Li et al 2022) contain high concentrations of Hinokione spathulenol, palmitic acid, α-linoleic acid, and linolenic acid. On the other hand, M. aegyptiaca L. essential oil is characterized by the abundance of dillapiole (Zouari et al 2011). Another study reported that linolic acid was one of the main compounds found in M. aegyptica L. extract, consistent with our findings (Shreshtha et al 2017). Fatty acids and hydrocarbons seem to constitute the main compounds of M. sylestries essential oil. Other species from the same genus were characterized by the abundance of oxygenated fatty acids, notably M. sherardiana, M. neglecta, and M. aegyptiaca L. (Adams et al 2007, Fadli et al 2012). The chemical compositions of the essential oils extracted from the Malva species displayed a large intra and interspecies variability, depending on the conditions under which the plants are grown. Essential oil chemical profiles might vary with seasons, soil composition, plant age, and geographical location (Delfine et al 2017, Hajdari et al 2016). Additionally, the plants' essence compounds depend on genetics and geographical location (Rajabi et al 2019).
(Table 1, 2) and (Fig. 8)
| NO | RT | Compounds | MW | Chemical formula | Content% |
|---|---|---|---|---|---|
| 1 | 16.667 | 5-Methyl-2-furaldehyde | 110.11 | C6H6O2 | 1.78 |
| 2 | 16.827 | Benzaldehyde | 106.12 | C7H6O | 1.85 |
| 3 | 17.209 | Trisulfide, dimethyl | 126.26 | C2H6S3 | 0.78 |
| 4 | 20.999 | Phenylacetaldehyde | 120.15 | C8H8O | 3.16 |
| 5 | 22.593 | Acetophenone, 2-hydroxy | 136.15 | C8H8O2 | 0.97 |
| 6 | 23.971 | Nonanal | 142.24 | C9H18O | 1.01 |
| 7 | 28.863 | Safranal | 150.21 | C10H14O | 1.15 |
| 8 | 29.177 | Cyclohexane, propadienyl | 122.21 | C9H14 | 1.36 |
| 9 | 30.418 | Coumaran | 120.06 | C8H8O | 2.08 |
| 10 | 31.023 | D-Carvone | 150.22 | C10H14O | 0.70 |
| 11 | 32.161 | α-Ethylidenbenzeneacetaldehyde | 146.18 | C10H10O | 1.78 |
| 12 | 33.310 | Limonen-10-ol | 152.23 | C10H16O | 1.00 |
| 13 | 34.201 | p-Vinylguaiacol | 150.17 | C9H10O2 | 6.40 |
| 14 | 36.562 | Benzeneacetaldehyde,α-(2 methylpropylidene) | 174.24 | C12H14O | 0.78 |
| 15 | 37.156 | β-Damascenone | 190.28 | C13H18O | 0.52 |
| 16 | 37.505 | 5-Amino-1-phenylpyrazole | 159.19 | C9H9N3 | 2.61 |
| 17 | 38.568 | Isodurene | 134.22 | C10H14 | 0.94 |
| 18 | 39.985 | cis-Geranylacetone | 194.31 | C13H22O | 0.80 |
| 19 | 40.505 | Tetradecamethylcycloheptasiloxane | 519.08 | C14H42O7Si7 | 1.01 |
| 20 | 41.334 | dehydroionone | 110.11 | C6H6O2 | 1.31 |
| 21 | 41.442 | β-E-Ionone | 192.29 | C13H20O | 3.32 |
| 22 | 41.593 | 6-Methyl-6-(5-methylfuran-2-yl) hept-2-one | 208.29 | C13H20O2 | 2.93 |
| 23 | 43.751 | Dihydroactinolide | 180.24 | C11H16O2 | 1.29 |
| 24 | 45.506 | Megastigmatrienone A | 190.14 | C13H18O | 2.00 |
| 25 | 52.290 | Myristic acid | 228.37 | C14H28O | 0.68 |
| 26 | 54.811 | Neophytadiene | 278.51 | C20H38 | 2.02 |
| 27 | 55.011 | Phytone | 448.6 | C24H48O7 | 6.64 |
| 28 | 55.662 | Hexacosane | 366.7 | C26H54 | 1.80 |
| 29 | 57.377 | 5-9-13-Pentadecatriene-2-one,6-10-14-trimethyl | 262.43 | C18H30O | 2.11 |
| 30 | 58.548 | Isophytol | 296.53 | C20H40O | 0.97 |
| 31 | 59.154 | Palmitic acid | 256.3 | C16H32O2 | 11.08 |
| 32 | 63.355 | Linolenic acid, methyl ester | 292.45 | C19H34O2 | 1.05 |
| 33 | 63.721 | phytol | 296.53 | C20H40O | 4.23 |
| 34 | 64.681 | Oleic acid | 282.47 | C18H34O2 | 18.05 |
| Carbohydrates Monoterpene Diterpene Fatty acids Others Non found Total (%) | 37.46 5.57 7.22 30.18 15.04 8.66 100 | ||||
Table 2. The chemical structures of the most representative phytochemicals in essential oil Mallow, a. p-Vinylguaiacol, b. phytone, c. Palmitic acid, d. phytol and e. Oleic acid.
In a supplementary study, the presence of PDB3 in the extract was examined using the HPLC technique, by comparing the retention time (tR) of the PDB3 standard chromatogram (Fig.S4) and the peak corresponding to the extract sample chromatogram. For the separation of the substances of the extract, the parameters of the column and the mobile phase were tested, as described above. In the isocratic elution mode, the elution time of PDB3 was around 10 minutes. As can be seen, the applied methodology is characterized by complete chromatographic separation of PDB3 from other components of the extract, which is a requirement for the effectiveness of the technique. The tR of PDB3 of M. sylvestris extract coincides with tR of the standard sample of PDB3 (Fig. S4). Given the chromatographic settings, the symmetry factor for the standard peaks of PDB3 was close to 1 with a tR of 10 minutes. Prodelphinidin is identified in cereals (Hordeum vulgare, Humulus lupulus), legumes (Pisum sativum L), fruits (Ribes rubrum, Sambucus nigra), Beverages and spices (Cinnamomum zeylanica L.) (Teixeira 2016). A study on Anacardium occidentale L. skin and flesh with a acetone/water ratio of 60/40 through the HPLC-DAD/ESI-MS method found prodelphinidin compounds (Michodjehoun et al 2009) The previous reports indicated the contents of vitamins, anthocyanin, saponin, tannins, alkaloids, flavonoids, and phenolic compounds (Hasimi et al 2017), which is consistent with our finding of PDB3 (tannin) composition.
3.5. Total flavonoids and total phenols assay
The total flavonoid and total phenol content were determined using the aluminum chloride colorimetric and Folin-Ciocalteu methods, respectively. To do this, the absorption spectra of the standard solution (10–50 ppm) of gallic acid and quercetin were recorded, and then the methanolic extract was analyzed (Fig. S5). The absorbance intensity of the solutions at their maximum wavelength was used for quantitative assays. A significant correlation was observed between absorbance and the concentration of gallic acid and quercetin for the analysis of flavonoids and total phenols in the extract, as shown by the excellent regression coefficients (R2 = 0.9684 and 0.9973). The ratio of flavonoids to phenols in the M. sylestries extract is presented in Table S2. The total phenol and flavonoid contents were found to be 97.267 mg gallic acid equivalent/g and 217.28 mg quercetin equivalent/g, respectively. These results indicate an acceptable content of flavonoids and phenols in the plant. The presence of these secondary metabolites suggests potential beneficial uses (Razavi et al 2011).
3.6. Antimicrobial activity
The disc-diffusion method was used to evaluate the qualitative antibacterial activities of M. sylvestris extracts. The methanol extract showed more pronounced effects against B. subtilis and E. coli with inhibition zones of 20 and 23 mm, respectively, while P. aeruginosa remained the most resistant bacteria with a diameter of 15 mm. In comparison, the aqueous extract of the plant exhibited more sensitivity toward B. subtilis than the other two strains (E. coli and P. aeruginosa) with inhibition zones of 21, 20, and 17 mm, respectively. Common antibiotics such as amoxicillin, erythromycin, cephalexin, and fluvoxamine contributed to the inhibition of all tested strains. Fluconazole showed the most sensitivity with an inhibition diameter of 38 mm, while cephalexin and amoxicillin were more resistant. Additionally, the methanolic extract of the M. sylvestris plant flowers provided high antibacterial effects against some human pathogens. Other studies also showed the potent activity of the flower extract of M. sylvestris against various bacteria strains. These results are consistent with our findings.
(Table 3)
| Bacteria | Zone of inhibition (mm) | |||||
|---|---|---|---|---|---|---|
| Methanolic extract (mg/disc) | Aqueous extract (mg/disc) | |||||
| 0.375 | 0.75 | 1.5 | 0.375 | 0.75 | 1.5 | |
| E. coli | 9.87 | 15.77 | 23.93 | 10.00 | 17.00 | 20.00 |
| P. aeruginosa | 9.00 | 11.30 | 15.47 | 14.00 | 18.00 | 21.00 |
| B. subtilis | 12.30 | 15.23 | 20.72 | 10.433 | 16.33 | 20.72 |
| a All tests were performed for three times and the data are the mean of three measurements. | ||||||
The present study focused on the green synthesis of silver/silver oxide nanoparticles and the chemical analysis of essential oil compounds of the M. sylvestris herb. Most of the medical effects of M. sylvestris have been attributed to the plant's secondary compounds. The essential oil of the plant contains 34 compounds, with major constituents including oleic acid, palmitic acid, phytone, p-Vinylguaiacol, and phytol. HPLC analysis revealed that the plant extract is a rich source of PDB3. The biosynthesized NPs were characterized using X-ray diffraction (XRD), Fourier transform infrared (FTIR), UV-visible spectroscopy, and scanning electron microscope (SEM). Besides, various techniques such as UV-Vis absorption and FT-IR spectroscopies confirmed the presence of flavonoid and phenolic compounds in the M. sylvestris extract, with gallic acid and quercetin being the most abundant. The antimicrobial influence of M. sylvestris extract varies depending on the type of microorganism, with Gram-positive bacteria being more sensitive than Gram-negative bacteria. Further research is needed to explore the extract's effects on other pathogenic microorganisms and its interaction with commonly used antibiotics to determine an appropriate dosage.
Ethical Approval
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Competing Interests
The authors have no relevant financial or non-financial interests to disclose.
Conflict of interest
The authors have no conflicts of interest to declare.
Funding
There is no any funding source for this publication.
Authors Contribution
The first draft of the manuscript was written, data interpretation by Ali Bekhradian. All authors contributed equally to the study conception and design of the revised article. Dr. Hamid Reza Rajabi and Prof. Bahador Karami to revise the manuscript and check all technical, grammatical mistakes in manuscript.
Data Availability Statement
The authors declare that all relevant data that support the findings of this study are available within the article. Additional data are available from the corresponding author upon reasonable request.
- Abdalameer NKh, Khalaph KA, Ali EM (2021) Ag/AgO nanoparticles: Green synthesis and investigation of their bacterial inhibition effects. Materialstoday: Proceeding 45: 5788-5792. https://doi.org/10.1016/j.matpr.2021.03.166
- Adams RP, (2007). Review of identification of essential oil components by gas chromatography/ mass spectrometry, 4th edition. J. Am. Soc. Mass Spectrom. 18: 803-806. http://dx.doi.org/10.1016/j.jasms.2007.01.001
- Amirhosseini ZK, Monsef R, Ehsanizadeh SA, Albahadly WKY, Majdi HSh, Amir AA, Dawood AH, Niasari MS (2023). Tailoring the photocatalytic activity of novel magnetically separable ZnFe12O19-chitosan bionanocomposites: A green preparation, structural characterization and comparative study. International journal of hydrogen energy. 48(95): 37286-37301.
- Atri A, Echabaane M, Bouzidi A, Harabi I, Soucase B M, Rafik Ben Chaabane R B, (2023). Green synthesis of copper oxide nanoparticles using Ephedra Alata plant extract and a study of their antifungal, antibacterial activity and photocatalytic performance under sunlight," Heliyon, vol. 9, p. 13484. https://doi.org/10.1016/j.heliyon.2023.e13484.
- Alharthi FA, Alghamdi AA, Zaqri NA, Alanazi HS, Alsyahi AA, Marghany AE, Ahmad N (2020) Facile one-pot green synthesis of Ag–ZnO Nanocomposites using potato peel and their Ag concentration dependent photocatalytic properties. Sci. Rep 10: 1-14. https://doi.org/10.1038/s41598-020-77426-y
- Camejo-Rodrigues J, Ascensão L, Bonet MÀ, Vallès J (2003). An ethnobotanical study of medicinal and aromatic plants in the Natural Park of ‘Serra de São Mamede’ (Portugal). J. Ethnopharmacol. 89: 199-209. http://DOI:10.1016/S0378-8741 (03)00270-8.
- Cecotti A, (2016). Chemical characterization of the volatiles of leaves and flowers from cultivated Malva sylvestris var. mauritiana and their antimicrobial activity against the aetiological agents of the European and American foulbrood of honeybees (Apis mellifera). Nat. Prod.11: 1527-1530. https://doi.org/10.1177/1934578X1601101026
- Chai WM, Shi Y, Feng HL, Qiu L, Zhou HCh, Deng ZW, Yan ChL, Chen QXi (2012). NMR, HPLC-ESI-MS, and MALDI-TOF MS analysis of condensed tannins from Delonix regia (Bojer ex Hook.) Raf and their bioactivities. J. Agric. Food Chem 60: 5013-5022. http://dx.doi.org/10.1021/jf300740d
- Delfine S, Marrelli M, Conforti F, Formisano C, Rigano D, Menichini F, Senatore F (2017) Variation of Malva sylvestris essential oil yield, chemical composition and biological activity in response to different environments across Southern Italy. Ind. Crops Prod. 98: 29-37. https://doi.org/10.1016/j.indcrop.2017.01.016
- Dowek S, FallahS, SalimiaRB, Jazzer M, Qawasmeh A (2020) Antibacterial, Antioxidant and Phytochemical Screening of Palestinian Mallow, Malva Sylvestris L. Int. J. Pharm. Pharm. Sci., 12: 12-16. http://dx.doi.org/10.22159/ijpps.2020v12i10.39053
- Eslami B, Ebrahimzadeh MA, Nabavi SM (2010) Biological activity of Mentha aquatica L. Pharmacologyonline. 2: 611-619.
- Fadli M (2012) Antibacterial activity of Thymus maroccanus and Thymus broussonetii essential oils against nosocomial infection – bacteria and their synergistic potential with antibiotics. Phytomedicine, 19: 464-471. doi: 10.1016/j.phymed.2011.12.003
- Gasparetto R (2012) Ethnobotanical and scientific aspects of Malva sylvestris L.: a millennial herbal medicine. J. Pharm. Pharmacol 64: 172-189. doi: 10.1111/j.2042-7158.2011.01383. x
- Goupy P (1999) Antioxidant composition and activity of barley (Hordeum vulgare) and malt extracts and of isolated phenolic compounds. J. Sci. Food Agric 79: 1625-1634. https://doi.org/10.1002/(SICI)1097-0010(199909)79:12%3C1625
- Hasimi, N, Ertaş A, Oral EV, Alkan H, Boğa M, Yılmaz MA, Yener I, Gazioğlu I, Özaslan C, Akdeniz M, Kolak U (2017) Chemical profile of Malva neglecta and Malvella sherardiana by LC-MS/MS, GC/MS and their anticholinesterase, antimicrobial and antioxidant properties with aflatoxin-contents. Marmara Pharm. J 21: 471-484. doi no: 10.12991/marupj.307461
- Hajdari CL, Mustafa B, Nebija D, Selimi H, Veselaj Z, Breznica P, Quave CL, Novak J (2016) Essential oil composition of Pinus peuce Griseb. Needles and twigs from two national parks of Kosovo. Sci. World J 5: 1-9. http://dx.doi.org/10.1155/2016/5393079
- Irfan A, Imran M, Khalid N, Hussain R, Raza Basra M A, Khaliq T, Shahzad M, Hussien M, Shah AT, Qayyum MA, Al-Sehemi AG, Assiri MA (2021) Phenolic and flavonoid contents in Malva sylvestris and exploration of active drugs as antioxidant and anti-COVID19 by quantum chemical and molecular docking studies. J. Saudi Chem. Soc. 25: 1-12. https://doi.org/10.1016/j.jscs.2021.101277
- Jain PK, Soni A, Jain P, Bhawsar J (2016) Phytochemical analysis of Mentha spicata plant extract using UV-VIS, FTIR and GC/MS technique. JOCPR 82: 1-6.
- Kuang Y, Chen Z, Megharaj M, Naidu R (2014) Heterogeneous Fenton-like oxidation of monochlorobenzene using green synthesis of iron nanoparticles. J Colloid Interface Sci 410: 67-73. doi: 10.1016/j.jcis.2013.08.020
- Karami M, Ghanbari M, Amiri O , Niasari MS (2020), Enhanced antibacterial activity and photocatalytic degradation of organic dyes under visible light using cesium lead iodide perovskite nanostructures prepared by hydrothermal method. Separation and Purification Technology. 253: 117526. https://doi.org/10.1016/j.seppur.2020.117526
- Patle TK, Shrivas K, Kurrey R, Upadhyay S, Jangde R, Chauhanet R (2020) Phytochemical screening and determination of phenolics and flavonoids in Dillenia pentagyna using UV–vis and FTIR spectroscopy. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 242: 118717. https://doi.org/10.1016/j.saa.2020.118717
- Leporatti ML, Ghedira K (2009) Comparative analysis of medicinal plants used in traditional medicine in Italy and Tunisia. J. Ethnobiol. Ethnomed 5: 1-8. http://doi: 10.1186/1746-4269-5-31
- Li X, Wang X, Zhao M, Zhang H, Liu Ch (2022) The Chemical and Pharmacological Research Progress on a Kind of Chinese Herbal Medicine, Fructus Malvae. Molecules 27: 5678. https://doi.org/10.3390%2Fmolecules27175678.
- Loizzo MR (2016) Edible flowers: a rich source of phytochemicals with antioxidant and hypoglycemic properties. J. Agric. Food Chem 64: 2467-2474. https://doi.org/10.1021/acs.jafc.5b03092
- Misak SN, Mohammed JA, Malik HJ (2011) Screening of antibacterial properties for some Iraqi plants against Salmonella typhimurium. Iraqi. J. Vet. Sci 35: 28-35. http://doi: 10.30539/ijvm
- Makabe H (2016) Studies on Prodelphinidins: Isolation, Synthesis, and Their Biological Activities, 1st ed., 48. Elsevier B.V.
- Machado S, Pinto S L, Grosso JP, Nouws HPA, Albergaria JT, Matos CD (2013) Green production of zero-valent iron nanoparticles using tree leaf extracts. Sci Total Environ 445-446: 1-8. doi: 10.1016/j.scitotenv.2012.12.033.
- Madhavi V, Prasad TNVKV, Reddy AVB, Reddy BR, Madhavi G (2013) Application of phytogenic zerovalent iron nanoparticles in the adsorption of hexavalent chromium," Spectrochim Acta A Mol Biomol Spectrosc, 116: 17-25. Doi:10.1016/j.saa.2013.06.045
- Mohani N, Ahmad M, Jahan M, Jahan N (2014) Evaluation of phytoconstituents of three plants Acorus calamus linn. Artemisia absinthium Linn and Bergenia himalaica boriss by FTIR spectroscopic analysis. Pak J Pharm Sci. 27: 2251-2255. PMID: 26045375.
- Mamta S, Jyoti S, (2012) Evalution of phytoconstituents of Acorus calamus by FT-IR and UV- VIS spectroscopic analysis. Int. J. Biol. Pharm. Res. 3: 498-501
- Michodjehoun-Mestres L (2009) Characterisation of highly polymerized prodelphinidins from skin and flesh of four cashew apple (Anacardium occidentale L.) genotypes. Food Chem 114: 989-995. https://doi.org/10.1016/j.foodchem.2008.10.052
- Manoukian, P, Melliou E, Liouni M, Magiatis P (2016) Identification and quantitation of benzoxazinoids in wheat malt beer by qNMR and GC–MS. LWT - Food Sci. Technol 65: 1133-1137. https://doi.org/10.1016/j.lwt.2015.10.014
- Nasiri S, Hosseinimehr SJ, Azadbakht M, Akbari J, Enayatifard R, AziziS (2015) Effect of Malva sylvestris cream on burn injury and wounds in rats. Avicenna J. Phytomed. 5: 341-354. 26909337.
- Oroumi Gh, Rozita Monsef R, Dawi EA, Aljeboree AM, Alubiady MHSh, Al-Ani AM, Niasari MS (2024) Achieving new insights on rational design and application of double perovskite Y2CrMnO6 nanostructures as potential materials for electrochemical hydrogen storage performance. Journal of Energy Storage. 85: 111161. https://doi.org/10.1016/j.est.2024.111161
- Parham S, Kharazi AZ, Bakhsheshirad HR, Nur H, Ismail AF, Sharif S, Krishna SR, Berto F (2020). Antioxidant, antimicrobial and antiviral properties of herbal materials. Antioxidants 9: 1039. http://DOI: 10.3390/antiox9121309.
- Paul D (2016) A review on biological activities of common Mallow (Malva sylvestris L.). Life Sci. 4: 1-5. https://journals.innovareacademics.in/index.php/ijls/article/view/14809
- Prudente AS (2013) Pre-clinical anti-inflammatory aspects of a cuisine and medicinal millennial herb: Malva sylvestris L. Food Chem. Toxicol 58: 324-331. DOI: 10.1016/j.fct.2013.04.042
- Quave CL, Plano LRW, Pantuso T, Bennett BC (2008) Effects of extracts from Italian medicinal plants on planktonic growth, biofilm formation and adherence of methicillin-resistant Staphylococcus aureus. J. Ethnopharmacol 118: 418428. https://doi.org/10.1016%2Fj.jep.2008.05.005
- Quave CL, Pieroni A, Bennett BC (2008) Dermatological remedies in the traditional pharmacopoeia of Vulture-Alto Bradano, inland southern Italy. J. Ethnobiol. Ethnomed 4: 1-10. http://doi: 10.1186/1746-4269-4-5
- Rajabi HR, Alvand ZM, Mirzaei A, Sajadiasl F (2021) Combination of plant mediated and sonochemical assisted synthesis for preparation of low-toxic cadmium selenide semiconductor nanoparticles: Study of the effect of extraction techniques, characterization, comparative study of biological activities. Surf. Interfaces 25: 101182. https://doi.org/10.1016/j.surfin.2021.101182
- Rajabi HR, Alvand ZM, Mirzaei A, Masoumiasl A (2019) Ultrasonic and microwave assisted extraction as rapid and efficient techniques for plant mediated synthesis of quantum dots: green synthesis, characterization of zinc telluride and comparison study of some biological activities. New J. Chem. 43: 15126-15138. https://doi.org/10.1039/C9NJ03144H
- Razavi GG, Zarrini SM, Molav G (2011) Bioactivity of Malva sylvestris L. a medicinal plant from Iran. Iran J Basic Med Sci. 14: 574-579. PMID: 23493458; PMCID: PMC3586856
- Siupp RA, Bailey JA (1977) The fungitoxicity of isoflavanoid phytoalexins measured using different types of bioassay. Physiol. Plant Pathol 11: 101-112. https://doi.org/10.1016/0048-4059(77)90093-5
- Savickiene N, Sikorska W, Musioł M, Janeczek H, Włodarczyk J, Pastusiak M, Gupta A, Radecka I, Parati M, Tylko G (2024) Efficacy of Proanthocyanidins from Pelargonium sidoides Root Extract in Reducing P. gingivalis Viability While Preserving Oral Commensal S. salivarius. Materials (Basel) 11: 1-15. https://doi.org/10.3390/ma11091499
- Surmaghi MHS (2011) Medicinal plants and phytotheraphy, The world.
- Shameli K, Ahmad MB, Zamanian A, Sangpour P, Shabanzadeh P, Abdollahi Y, Zargar M (2012) Green biosynthesis of silver nanoparticles using Curcuma longa tuber powder. Int J Nanomedicine 7: 5603-5610. https://doi.org/10.2147/IJN.S36786.
- Slathia S, Gupta T, Chauhan RP (2021) Green synthesis of Ag–ZnO nanocomposite using Azadirachta indica leaf extract exhibiting excellent optical and electrical properties," Physica B: Physics of Condensed Matter 621: 413287. http://dx.doi.org/10.1016/j.physb.2021.413287
- Shreshtha S, Shreshtha Sh, Anushi J, Ambika J, Nitesh J, Anupmaet H (2017) Study of total phenol, flavonoid contents and phytochemical screening of methanolic crude extracts of two weed plants. Annals of Plant Sciences, 6: 1645-1648. http://dx.doi.org/10.21746/aps.2017.06.004
- Teixeira N, Mateus N, Freitas V (2016) Experimental data for the synthesis of a new dimeric prodelphinidin gallate. Data in Brief 85: 631-636. http://dx.doi.org/10.1016/j.dib.2016.06.023
- Thériault M, Caillet S, Kermasha S, Lacroix M (2006) Antioxidant, antiradical and antimutagenic activities of phenolic compounds present in maple products. Food Chem 98: 490-501. http://dx.doi.org/10.1016/j.foodchem.2005.05.079
- Walter S, (2015). Plant Systematics, Fourth Edi. Oxford: Sinauer Associates.
- Williams GI, Owen EA, (1954). A low-temperature X-ray camera. Journal of Scientific Instruments, 31: 49 – 54.
- Williamson GK, Hall WH (1953). Discussion of the Theories of Line Broadening. Acta Metall. 1: 22.
- Yallappa S, Manjanna J, Dhananjaya BL (2015) Phytosynthesis of stable Au, Ag and Au–Ag alloy nanoparticles using J. Sambac leaves extract, and their enhanced antimicrobial activity in presence of organic antimicrobials. Spectrochimica acta part a molecular and biomolecular spectroscopy 137: 236-243. https://doi.org/10.1016/j.saa.2014.08.030
- Zouari N, Fakhfakh N, Zouari S, Sellami M, Abid M, Ayadi MA, Zaidi S, Neffati M (2011) Volatile and lipid analyses by gas chromatography/mass spectrometry and nutraceutical potential of edible wild Malva aegyptiaca L. (Malvaceae). Int. J. Food Sci. Nutr 62: 600-608. https://doi.org/10.3109/09637486.2011.564157