Green Synthesis by Bacteria
Green Synthesis by Fungi
Green Synthesis by Algae
Green Synthesis by plant extracts
Short review on Phyllanthus emblica, Psidium guajava and Lawsoniainermis.
A. Phyllanthus emblica
B. Psidium guajava
Part 1. Screening of selected plant extracts for green synthesis of silver nanoparticles (AgNPs):
Analysis and characterization of the produced AgNPs
Chapter 1: Chromatographic investigation of the ethyl acetate fraction of emblica:
Identification of compound E1:
Identification of compound E2 and E3:
Identification of compound E4:
Identification of compound E5:
Chapter 2: Chromatographic investigation of ethyl acetate fraction of Psidium guajava leaves
Identification of compound G1:
Identification of compound G2:
Identification of compound G3:
Identification of compound G4:
Identification of compound G5:
Chapter 3: Chromatographic investigation of ethyl acetate fraction of Lawsonia inermis leaves
Identification of compound H1:
Identification of compound H2:
Identification of compound H3:
Compound E1 (Trans -Cinnamic acid):
Compound E2 (Methyl gallate):
Compound E3 (Gallic acid):
Compound E4 (Emblifatmin):
Compound E5 & G5 (Quercetin-3- O -α-arabinofuranoside):
Compound G1 (Pyrogallol):
Compound G2 (Quercetin):
Compound G3 (Quercetin-3- O -β-xylopyranoside):
Compound G4 (Quercetin-3- O -β-arabinopyranoside):
Compound H1 (P -Coumaric acid):
Compound H2 (Lawson):
Compound H3 (Luteolin):
Metal nanoparticles synthesis is a leading topic of research in modern material science owing to their distinctive potential applications in the field of electronic, optoelectronic, information storage and Health care. Among the all noble metal nanoparticles, silver nanoparticles are onethe main products in the field of nanotechnology which has acquired limitless attentiondue to their unique properties such as chemical stability, good conductivity, catalytic and most important antibacterial, antiviral and antifungal activities.Nevertheless, there is still need for economic commercially viable as well as environmentally clean synthesis route to synthesize the silver nanoparticles.
Mostly, nanoparticles are prepared by different kinds of chemical and physical methods which are quite expensive and potentially hazardous to the environment which involve the use of toxic and unhealthy chemicals that are responsible for various biological risks.There aretwo main approaches which are involved in the synthesis of silvernanoparticles, either from bottom to top (or bottom up) approach or a top to bottom (or top down) approach.
In bottom to topmethod,nanoparticles can be synthesized using chemical and biologicalmethods by self-assemble (aggregation) of atoms to form a new nuclei which will growinto a particle withinnano-sizes. While in top to bottommethods, suitable bulk material break down intofine particles by size reduction with different techniques likegrinding andmilling.
Methods of silver nanoparticles synthesis:
Silver nanoparticles are produced using a variety of methods. In environmental maintenance, there is a need to develop eco-friendly procedures to avoid the toxic chemicals which cause undesired adverse effect in medical applications. Hence, researchers inspired on biological systems to develop benign nanoparticles using microorganisms (bacteria, fungi and algae) and plant extracts which further termed as green synthesis approaches.
Among the different biological methods of silver nanoparticle synthesis, microbe-mediated synthesis is not of industrial feasibility due to the need of highly sterile conditions and their maintenance. So that, the use of plant extracts for this purpose ispotentially valuable over microorganisms due to the easeof development and absence of the complicated process ofmaintaining and preserving the cell cultures. Many researchers had developed AgNPs using different plant extracts such as Cinnamomumcamphora, Aloe vera, Coriandrum sativum, Phyllanthus amarus and Rose leaf.
According to the order and the aim of the work, this study involved three parts as follows:
Part I : Screening of plant extracts for green synthesis of silver nanoparticles (AgNPs):
Forty two medicinal plants were collected depending on their availability on Egypt. These plant were extracted and screened for their activity towards green synthesis of AgNPs. A new modified method was used in order to screen large number of extracts in short time (i.e. high throughput screening). In this method, the synthesized nanoparticles were evaluated in using 96-well plate which is transparent to facilitate the color monitoring and easy to be read spectrophotometrically using microplate reader.The green synthesis activity by the different plant extracts was monitored and evaluated by color change and UV-Vis absorption at time intervals.
Out of these 42 plant, only 3 plants viz., Pyllanthusemblica fruits, Psidium guajava leaves and Lawsoniainermis leaves, showed high AgNPs synthetic activity. The most active plant extracts were further partitioned with ethyl acetate. The results showed thatthe EtOAc fraction of the three active plants showed superior AgNPs green synthesis over their remaining aqueous. Thus, chromatographic columns were used to isolate the active compounds from the EtOAc fraction of each plant.
Detection and characterization of the produced AgNPs:
Subsequently, the produced AgNPs were subjected to intensive analysis to detect and characterize the produced nanoparticles. The color changeof the silver ion solution is the fast indicator in monitoring the synthesis of AgNPs. Silver nanoparticles have a well-known yellowish-brown color which is easily monitored when compared to the original light yellow color of the plant extract. This color was formed due to excitation of Surface Plasmon Resonance phenomena. Beside the color change, UV-Vis spectra of the reaction mixture confirmed the synthesis of the AgNPs. The nanoparticles produced by the EtOAc fraction of the three plant extracts displayed absorption peak appeared at 440 to 460 nm. These peaks are corresponding to the characteristic Surface Plasmon Resonance of the resulting AgNPs.
FTIR analysis was carried out in order to identify the possible functional groups in the biomolecules which may be responsible for reducing the silver ions. FTIR spectra of P. emblica, P. guajava and L. inermis extracts showed bands at 1034, 1384, 1637 and 3447cm-1.These bands are representing the C-O stretching vibration, aliphatic CH2/CH3groups, carbonyl groups and stretching vibrations of alcoholic and phenolic O-Hgroups, respectively.
Transmission electron microscopy was used to detect and characterize both the size and the morphology of the formed AgNPsusing the three active plant extracts. TEM micrographs showed that the produced AgNPs were mostly spherical, poly-dispersed with size range 5-30 nm and are surrounded by a thin layer of the extract.
The green synthesized AgNPs of the three plants were biologically evaluated towards both cytotoxicactivity towards human cancer cell lines and antioxidant activity. The cytotoxic activity of AgNPs was compared to that of their extracts against two mammalian cancer cell lines; colon cancer cells (HCT-116) and breast cancer cells (MCF-7). Cisplatin (anticancer drug) showed IC5010000 and 5000 nM for MCF-7 and HCT-116 cell lines, respectively. The AgNPs synthesized using P. guajava leavesshowed nearly the same activity that produced by the plant extract alone although the cytotoxic activity was decreased in case of AgNPs synthesized using P. emblica than that of its plant extract. While, the AgNPs synthesized using L. inermis leaves extract showed an increased cytotoxic effect against both breast cancer (MCF-7) and colon cancer (HCT-116) cell lines when compared to its plant extract. This increase in the cytotoxic activity may be due to a synergetic effect of both AgNPs and these phytocomponents present in henna leaves.
Moreover, the three plant extracts showed nearly a comparable activity to vitamin C (positive control) using ABTS superoxide antioxidant assay. That may be attributed to the polyphenolic content of the tested extracts. While the AgNPs synthesized using P. emblica, P. guajava and L. inermis showed a very close activity to each otherbut less active that their extracts alone. This may explainthat the activity is mainly due to the AgNPs and not due to the components in each plant extract. Where, the antioxidant molecules involved in the reduction process of the Ag+ ions are no more active as ABTS+ free radical scavengers after their oxidation. These results may also emphasize the role of these polyphenolic compounds in AgNPs synthesis.
Part II :Chromatographic separation of the phytochemical components with green synthesis activity:
The ethyl acetate extracts of the three plants viz.,emblicafruits, guava leaves and Hennaleaves were further subjected to chromatographic separation in order to isolate, purify and detect the nano-active molecules present in the plants. The separation process was done using silica gel column chromatography followed by purification of the collected fraction using SephadexLH-20. The isolated compounds were detected and characterized using physical, chemical and different spectral analysis.
The ethyl acetate fraction of P. emblica yielded five compounds including four known compounds that have been previously isolated from the plant. These compounds are; trans -cinnamic acid (E1), methyl gallate (E2), gallic acid (E3) and quercetin-3- O -α-arabinofuranoside (avicularin) (E5). Beside these compounds, emblifatmin (E4) a new compound was isolated for the first time from nature. This new compound (E4) was characterized using FTIR, 1H-NMR, 13C-NMR, HSQC, HMBC and high resolution mass.
Also, five known compounds were isolated from the ethyl acetate fraction of the P. guajava leaves extract. Quercetin-3- O -β-arabinopyranoside(G4) was isolated for the first time from this plant. While the other four compounds viz., pyrogallol (G1), quercetin (G2), quercetin-3- O -β-D-xylopyranoside (reynoutrin) (G3) and quercetin-3- O -α-L-arabinofuranoside (avicularin) (G5) were previously reported from guava leaves.
Meanwhile, three known compounds were identified from the ethyl acetate fraction of L. inermis leaves extract. Firstly, lawsone (H2) that was detected in the EtOAcfraction using co-chromatography technique. The other two compounds were p -coumaric acid (H1) and luteolin (H3). It worth to note that the isolation of these three compounds was reported before from henna leaves.
Part III :Activity of the isolated compounds towards the green synthesis of AgNPs:
In this part, the thirteen pure compounds isolated from the active plant extracts viz.,emblicafruits, guava leaves and hennaleaves were investigated for their AgNPs green synthesis activity. The results showed that four compounds isolated from P. emblica were active towards the green synthesis process.
- Gallic acid (E3) and methyl gallate (E2) showed high activity with slightly superiority of gallic acid.
- Emblifatmin (E4) exhibited green synthesis activity however, it was lower than that of E2 & E3 based oncomparing color change intensity and UV absorption
- Avicularin (E5) showed lower activity in AgNPs green synthesis when compared to the other active compounds isolated from the emblica.
All these four compounds may contribute to the green synthesis activity of emblica. However, compound E1 was completely inactive towards the green synthesis of AgNPs.
Moreover, all compounds isolated from P. guajava plant extract were active but with some difference in the activity.
- Pyrogallol (G1) was the most active compound which may strongly contribute to the green synthesis activity of guava.
- Quercetin (G2) was less active towards the green synthesis from compound G1 but it was more active than the other quercetin glycosides isolated from guava.
- Compounds G3 - G5 (quercetin glycosides) were in a relatively close activity to each other which may indicate that glycone part is not involved in the green synthesis process.
- Furthermore, two compounds were active from the isolated pure compounds from henna leaves. These compounds are lawsone (H2)and luteolin (H3) which thought to be contributed in the AgNPs green synthesis activity of henna leaves extract.
- Compound H1 (p -coumaric acid) was completely inactive confirming that there is no contribution from H1 in the green synthesis activity of henna.
From above results, we can conclude that compounds with galloyl moiety are more active than other compounds having flavonoid nucleus towards the green synthesis of AgNPs.
Natural products and its contribution in the medicine:
Natural forests are precious houses of phytochemicals and they never lose their identity. World Health Organization reports refer to, in some parts of primary health care, about eighty percent of the world's population depend on traditional medicinal system (Bonigala et al., 2016). Three hundred years ago, synthetic drugs had no exist yet, only plants and their extracts were the prime source of medicine over all the world. Today, there are many drugs are available in the market which is depend on the traditional remedies as well the ethnopharmacological studies. For example, commercially medicine such as morphine, ergometrine, digitalis, quinine and atropine are most wanted in the medicinal market till now. In this scenario science has been developed, the nutrients and chemicals founded in the plant extracts are easily drilled in scientific testing. There are many plant extracts are used nowadays in the manufacture of some cosmetics, shampoos, soaps, food flavoring agents and medicines. The secondary metabolites present in the plant extracts like alkaloids, glycosides, phenols, terpenoids and volatiles oils are used in numerous active drugs. In the present decade, there is a considerable scientific and commercial interest in the discovery of new anticancer agents from natural resources. More than 50% of all drugs in medical research and clinical trials for anticancer activity are originated from natural sources (Bonigala et al., 2016). Different plants extracts has been shown anticancer activity. Vincristine and Vinblastine from Catharanthus roseus, Taxol and Docetaxel from Taxus brevifolia and Camptothecins from Camptotheca acuminate are considered to be the best potential anticancer agents derived from plants (Prakash et al., 2013).
Nanobiotechnology, is a science deals with the synthesis of nanoparticles and their application in various fields. Natural products have showed wide contributions in the nanotechnology field. One of these applications is involving in the synthesis and stabilization of nanoparticles. The biological synthesis of metal nanoparticles is considered as one of the important areas of nanotechnology. The biological synthesis of metal nanoparticles by using the plant extracts is called green synthesis, which is safe and ecofriendly in nature. It needs less time, less technology, low cost and causes no health hazards when compared to other metal nanoparticles synthesis methods such as physical and chemical methods (Iravani et al., 2014). Green synthesis with plants is an advantageous technique than other biological methods i.e. synthesis of nanoparticles using microbial extracts needs complicated procedures in bacterial and fungal culture maintenance (Kalishwaralal et al., 2010). Among green synthesized metal nanoparticles, the silver nanoparticles are the most widely used in nanoscience due to its antimicrobial activity. In pure form, silver is highly toxic to microbes and when it is converted to its nano-level, the activity increases several times . From another point of view, reason of accepting the silver nanoparticles throughout the world is that silver is less toxic to humans than other metals and when tested for curing of various diseases it lefts no allergic reactions (Bonigala et al., 2016). Many researchers had developed silver nanoparticles using different plant extracts such as Cinnamomumcamphora, Aloe vera, Coriandrum sativum and Rose leaf (Abdelghany et al., 2017).
Medical treatments significantly are present in numerous Egyptian medicinal plants. To treat many diseases in ancient Egypt, a lot of different plant species were used, e.g. diabetes, skin diseases, liver function disorders, respiratory and nervous systems. From this plants, the most frequently used are: Nerium olender, Hyoscyamusaureaus, Peganum harmala, and Citrulluscolocynthis (El-Demerdash, 2001).
Aim of the work
In the present study, we aim to investigate some commonly known Egyptian medicinal plantsfor their ability to green synthesize silver nanoparticles from aqueous solution of AgNO3. The produced silver nanoparticles will be separated, identified and characterized.Characterization of the formed nanoparticles will be achieved by:
- UV-Vis spectroscopy: depending on surface plasmon resonance peaks.
- FTIR spectroscopy: to detect the active functional groups.
- Transmission electron microscopy (TEM): to detect the shape as well as the size of the nanoparticles.
The isolated capped silver nanoparticles will be further evaluated for their anticancer activity against MCF-7and HCT-116 cell lines using MTT assay. Moreover, the antioxidant activity for the isolated silver nanoparticles will be investigated using ABTS●+ assay.
The most active plant extracts towards the green synthesis of silver nanoparticles will be subjected to chromatographic isolation to identify and determine the bioactive molecules present in these plants and are responsible for the green synthesis activity.
Sustainable and green chemistry simply is just a different routes of study and rational how chemistry and chemical manufacturing can be achieved. Throughout the decades,variousbasics have been suggested to be used when consider about the strategy, development and applications of chemical outputandprocedures. These philosophiesempower researchers to protect and benefit the economy, people and our planet by finding creative and pioneer ways to decrease waste, preserve energy, and discover substitutes for hazardous substances(Clark, 1999).
Green chemistry and Nanotechnology:
Nanotechnology is a very important arena of recentresearches dealingwith planning, designing, production and handlingvery small particlestructures which its size ranging from roughly 1 to 100 nanometer.Inthe mentioned size range, almost all the properties (such as chemical, physicaland biological) varied in substantialbehaviors of both theatoms or molecules and their corresponding large bodies. Innovativeapplications of different nanoparticles and nanomaterials are risingquickly on severalaspectsbecause of their totally new orimproved properties depended ontheir sizes, their shapeandthe distribution pattern.It is rapidlybeinginnovative in a huge number ofarenaslikemedicine, food, health care, space manufacturing,environment, mechatronics,optical science, , electronicalproducts, chemical industries, energyfields, catalysis, light producers, cosmetics,nonlinear and linear optical apparatus and electro-chemical applications.Great development in these growing technologieshad revealedusefulfrontiers. The synthesisof small nanoscalematerials are includes then, inevaluation or usage of their ambiguousphysico-chemical properties(Kaviya et al., 2011a).
Green Nanotechnology considers as a new platform todesign innovative products that are generous to human and environment health and has massive potential to revolutionize bulky scalenano-synthesis procedures. These green synthesis methods for nano-materials are assumed to improve environmental and biomedicalsegments of nanotechnology implementations in the future.Although greennanotechnology displays a luminous picture of clean, environmental friendly and safe future, it has the challenge to not only deal with the probable toxicity matters but to make a new ground for maintainablenano-materials production with bearing in mindthe environmental and healthaspects. The value of nano-materials createdby green assemblyway is similar to their chemical analogues and one canchange the produced nano-materials properties by directing the reaction conditions(Joshi, 2016).
Nevertheless nanoparticles are used for the different previously mentioned purposes, mostly metallic nanoparticles are well-thoughtto be the most promising.They exhibitnotable antibacterial properties because of their huge surface area to volume ratio, which is of attention for researchers owing to the rising microbial resistance towards the metal ions, antibiotics and the emersion of resistant strains(Khalil et al., 2013).
Among the all noble metal nanoparticles, silver nanoparticlesare prime productsin the arena of nanotechnology which has acquired unlimited interests owing to their exceptional properties such as chemical stability, perfect conductivity, catalytic and most important bactericidal, antiviral, fungicidal in addition to anti-inflammatory activities which may be useful in composite fibers, cryogenic super-conducting materials, cosmetic products, food production and electronic apparatuses(Klaus-Joerger et al., 2001).
In bio-medical purposes; silver nanoparticles could be usedin wounddressings, skin creams and disinfectantformulas, the Agused as an microbial and it has a widerange biocidal activitytowards many microorganisms, this action is done bydestroying theircellularmembrane which resulting in disrupting the cell functionality.
Synthesis approaches of silver nanoparticles (AgNPs):
Generally, nanoparticles are synthesizedusing different kinds of chemical or physical approachesthat are relativelyin high cost and possiblydangerous to environment. These methodsincludeusage of poisonous and unhealthy chemicals whichmay causenumeroushazards to the humans. The advancement of new biological dependentmethods for the synthesis of nanoscale particleshas been developed intoan vitaldivision of nanotechnology.
Generally, there are2princibles which are elaborated in the production of silvernanoparticles, first approach is thebottom to top (or bottom up)methodand the second is atop to bottom (or top down)method(Figure1& 2).
Firstly, in thebottom to topmethod,the nanoscale particles could be producedby means of chemicalsor biological process by self-accumulate of atoms to form a new nuclei that furtherraise its size into a particle within the nanoscale as shown in Figure1. Secondly, in top to bottom method, appropriatelargeparticlesmashed down into smallfine pieces by reducing the sizeusingnumerous lithographic procedureslikegrinding, crushing &milling.
Abbildung in dieser Leseprobe nicht enthalten
Figure1. Different approaches of synthesis of silver nanoparticles (Author’s own work).
Abbildung in dieser Leseprobe nicht enthalten
Figure2. Illustration of the differences between top to bottom approach and bottom to top approach, (Nirmohi and Agnihotri, 2016).
In bottom to top approach, chemically reduction process is the mostcommon method to synthesize thesilver nanoparticles(Hurst et al., 2006).
-arious organic and inorganic chemicalshave a reducing power, likesodium borohydride (NaBH4), sodium citrate, DMF, elementalhydrogen, ascorbateand polyethylene glycol (PEG)typically usedfor silver metal ions (Ag+)reduction in aqueous media(Iravani et al., 2014).
For sizemaintenance of the nanoparticles,capping agents may be used. A unique advantageof this technique is that a greatamount of nanoparticlescould be produced in a relative short time. Using this kindof nano-production; many chemicals used are considered to be toxic which will lead to environmentally harmfulbyproducts. It could be the causethatguided to the biosynthesis of nanoscale particles througheco-friendlymethod that includes noutilization oftoxicmaterialsconsequentlyshowedto become agrowing imperiousdemand to proceedeco-friendly procedures. So, the advancement of green synthesis of nanoparticlesis progressing as a remarkablesection of nanotechnology; which the biological entities like microorganisms are being use in, also the usage of plant extractin the production of nanoparticles may becomean alternative to chemical and physical methods in an ecofriendlymanner(Ahmed et al., 2016).
On the other hand, in top to bottom method,the nanoscale particles are mostlyassembled by evaporation then condensation through a tube furnacein the atmospheric pressure. Using this technique, the base material in a boat like place, centered at the furnace where it is vaporizedinto a transporter (carrier) gas. silver, gold and lead sulfide nanoparticlesreported to have been already synthesized using the evaporationandcondensationmethod.
The production of AgNPs byusing a tube furnace has severaldisadvantages as it take upa bulky space and devour a hugeamount of energy while increasingthe environmental temp.around the source material,and it also requires a muchtime in order toachieve thermal stability(Prathna et al., 2011). Besides; the standard tube furnace needs powerusing up of more than plenty of kilowatts and a preheating timeof some minutes to attain a stable operating temperature.This technique has many major drawbacks, one of them is the limitations found in the surface structure of the produced nanoparticleswhile the majorphysical properties of nanoparticles are highly reliant onthe surface shape and structure in indication to surface chemistry.
Generally, whatever the method used, it is concluded that the chemical approaches have certain drawbacks with them either in chemical contamination throughout their synthesis procedures or later in applications.
Biological Methods of Green Synthesis of AgNPs:
The privilegeof green synthesis methodwhen compared to chemical and physical methods is being eco- friendly, lower cost and it can be expanded for larger scale synthesis of nanoparticleseasily. Moreover, it does not need high temperature using, pressure, energy or toxic chemicals (Dhuper et al., 2012). Literature have reported synthesis of AgNPsby biological method using micro-organisms like(bacteria, fungi) and plants,owing to their antioxidant properties and their reducing powerwhich is responsible of reducingthemetal ion solutionsto their corresponding nanoparticles. From the several biological approaches of AgNPsproduction, synthesis using microbesis not of industrial possibilitybecause of therequirements of extensivesterile conditions and their conservation.Thus, usage of plant extracts in that purpose isactuallybeneficialthan the usage of microorganisms due to the easeof improvement, the low bio-hazard and complicatedmethods ofmaintaining cell cultures(Kalishwaralal et al., 2010). Using plants is consideredas one the best approach for productionof nanoparticles,as it has no toxic chemical substancesalong withproviding natural capping (covering) agents which help in stabilization of the synthesized nanoparticles. Furthermore, usage of the plant extracts also diminish thehigh cost of microorganismsisolation and their important maintenance culture media. This enhances the price competitive possibility of nanoparticles synthesized by plants over that synthesized by microorganisms.
Green Synthesis by Bacteria
Husain et al. (2015) studied the biologically production of silver nanoscale particles using thirty cyanobacteria, thecyanobacterial aqueous extracts were subjected to AgNPsproduction at 30 °C. By screening of these aqueous extracts which contain AgNPs in UV-Vis range, theydisplayeda single peak indicating the formation of AgNPs. Scanning electron microscope micrographs of AgNPsproduced by the cyanobacterial extracts exhibited that, although the production of AgNPshappenedby all tested strains, at differenttime span of the(30 to 360 hour), the resulted morphologyas well the size(38-88 nanometer) of the nanoparticleswere different(Husain et al., 2015).
The biological synthesis ofAgNPs using the extracellularextract of P. aeruginosa (Melisa A. Quinteros et al., 2016) and E. coli (Divya et al., 2016)wasreported. Then, the produced silver nanoparticles were evaluated for their antimicrobial activity against human pathogens viz., S. aureus, S. epidermidis, Proteus mirabilis, Enterococcus faecalis, Acinetobacter baumannii, Klebsiella pneumoniae, P. aeruginosa,Bacillus subtilis, S. typhi, E. coli and Vibrio cholerae.
Antimicrobial activity of AgNPs biosynthesized using bacteria was tested for their synergisticactivity with antibiotics towardshuman pathogenic microorganisms(Divya et al., 2016). The action mechanism of the synthesized silver nanoparticles and their link to the production of oxidative stress inside bacterial cells was investigated.AgNPs generated oxidative stress was demonstrated by (M. A. Quinteros et al., 2016) in P. aeruginosa, E. coli, and S. aureus resulted by the increasing of reactive O•species and thisresulted inbetter-quality antimicrobialactivity.
The production, detection,and investigation of the antimicrobial activity of silver nanoparticlesusingnatural isolated extract of Corynebacterium glutamicum wasdemonstrated (Gowramma et al., 2015), which resulted with nanoparticlesits size rangedaround 15 nanometer. These produced AgNPspossessed anpromotedantibacterial activity towardssome pathogenic species.AgNPssynthesized using Bacillus thuringiensis and Enterobacter cloacae were of spherical shape, amorphous with sizes under 100 nanometerand showed much improvedwound healing activitywhen compared to the control groups by using the histological analysis (Pourali et al., 2016).
Bacilluspumilus, Bacilluslicheniformis, and Bacilluspersicus showed high capability in catalyzing the production of silver nanoparticles,producingnanoparticles ranging in size between 77 to 92 nanometer. The synthesizedsilver nanoparticles, especiallythose preoduced by B. licheniformis, displayedmuch stability and exhibitedan outstanding in vitro microbicidalactiontowardsdifferenthuman pathogens, beside that theyshowed a significant antiviral activity towards the Bean Yellow Mosaic Virus(Elbeshehy et al., 2015). Synthesis of AgNPsExtracellularlywas also reported by P. columellifera subspecies Pallida. They showed in vitro fungicidal activity towards superficial mycoses caused by fungal infection including Trichophyton rubrum, Candida tropicalis, C. albicans and Malassezia furfur (Anasane et al., 2016).
Green Synthesis by Fungi
Fungi as well as other microorganisms considered asa useful candidates forbioproduction of metallic nanoscale particles with various size ranges, due to their capacity to producegreatquantity ofenzymes and numerous reducing agents. Biological synthesisof AgNPs was found to be around 100 nm using the fungus Pestaloptiopsispauciseta, and 20–80 nm using Candida albicans (Rahimi et al., 2016).
Endophytic fungus Fusarium species was tested forbiologically synthesis of AgNPs extracellularly, and thetransmission electron microscopyconfirmed the synthesis of little size range and spherical AgNPswith rangeof 15 to 20 nanometer. Furthermore, the antimicrobial effect of the produced AgNPs was examined towards Salmonella typhi, S. aureus, and E. coli which showed maximum inhibition zones 26, 26, and 28 mm, respectively, by using 60 µL fromthe synthesized AgNPs(Singh et al., 2015).
Moreover, silver nanoparticles are biologically synthesized extracellularly by the mold of Fusariumgraminearum resulting in nanoparticles withsizeaverage about 45 nanometer and showed antimicrobial effect on Pseudomonas aeruginosa, C. albicans, and E. coli micro-organisms (Shafiq et al., 2016). Also (El-aziz et al., 2015) demonstrated that the production of AgNPsis safe and economically useful by successfully synthesized cultures of Fusariumsolani with average size from 5 to 30 nanometrer and high stability.
Three endophytes(fungi), Penicilliumochrochloron PFR8, Aspergillusniger PFR6, and Aspergillustamarii PFL2, wereobtained from Potentillafulgens L. medicinal plant and they were used for the biological synthesis of silver nanoparticles.The nanoparticles synthesized using the fungus A. tamarii PFL2 showed the lowest average of nanoparticle size(3.5 nanometer)when compared withother produced AgNPs synthesizedby the other two fungi P. ochrochloron and A. niger,whichgeneratednanoparticles with size averagesaround 8.7 and 7.7 nanometer, respectively (Devi and Joshi, 2015).
Silver nanoparticles produced by Trichodermaviride showed size rangeof 20 to 50 nanometer. The biosynthesizedAgNPs significantly inhibited of the growth of all tested pathogenic bacteria (Elgorban et al., 2016). Biological synthesis of AgNPsby extracellular isolate of Aspergillus versicolor ENT7 (has potential reducing power) has been noted and displayed a promising antioxidant and antibacterialeffect. Different Aspergillus specieswere screened for AgNPs synthesis ability, Aspergillus fumigatus was the besteffective species exhibited the highest reductioneffectbetween the investigated species.However, Aspergillusflavus displayed the smallestactivity in the biosynthesis of nanoparticlesthat was rational with its low nitrate reductase activity. The produced AgNPsshowed size ranges of 25, 45, 45, and 65nm synthesized by A. niger, A. fumigatus, A. clavatus and A. flavus, respectively (Elgorban et al., 2016).
Saxena et al, used plant endophyte fungi Sclerotiniasclerotiorum MTCC 8785strain inbiosynthesis of silver nanoparticlesand investigation of antibacterial properties of the synthesized nanoparticles. TEM images showed that the nanoparticles are spherical in shape and average size rangeof nanoparticles from 25-30 nanometer(Saxena et al., 2016).
Another study reported that A. niger also was used for biological synthesis of silver nanoparticlesdisplaying size range from 11 to 35 nanometer(Mohammed, 2015).
Myco-synthesis producingsilver nanoparticles with average size of 40 nanometer was successfully carried out by endophytic Colletotrichum species. The Escherichia coli DNA investigated with the AgNPsbiosynthesized using Colletotrichum speciesdisplayed deformed and destroyed deoxyribonucleicacid demonstrating the action of AgNPs(Azmath et al., 2016).
Green Synthesis by Algae
Synthesis process noted to besignificantlyquick, and AgNPs with size of 33 nanometer were produced withinfew minutes from silver solutions (Ag+) contactedwith the isolate solution of Pithophoraoedogonia algae (Sinha et al., 2015). While , nanoparticlessynthesizedusingother strains of micro-algae including Limnothix sp., Spirulina sp.,and Botryococcusbraunii showed size average of25, 13,and 15nm, respectively(Patel et al., 2015).
-arioussilver nanoparticles concentrations biologically synthesized by Anabaena oryzae, Calothrixmarchic, and Nostocmuscorum were evaluated its antitumor efficiency against in vitro EhrlichAscites Carcinomaassay (Abdelghany et al., 2017). Furthermore, theaqueous isolate extract of Amphiroafragilissima was used in reduction process ofAgNPssynthesis. The produced nanoparticlesshowed significant antibacterialeffecttowards E. coli, K. pneumoniae, S. aureus, and P. aeruginosa. Also, these producedAgNPsby using aqueous extract of algae Caulerparacemosa displayeda significantly antibacterial activity(Kathiraven et al., 2015).
Laurenciaaldingensis and Laurenciella species . algae aqueous isolateshave been used to biosynthesize silver nanoparticles from silver ion solution. The produced AgNPsexhibited high toxicities on different human cell lines(Vieira et al., 2016). While, biological synthesis of AgNPs using Spirulinaplatensis with size range around 11 nanometer and Nostoc sp. with size range of most particles was about 20nanometer atroom temperature was studied. The produced nanoparticlesexhibited apromisingantimicrobialeffect towards differenthuman pathogenic organismssuch as Staphylococcus aureus, K. pneumoniae, and Escherichia coli (Abdelghany et al., 2017).
Also, AgNPsbiologically synthesized usingpowder algal biomass of S. platensis, the synthesized nanoparticleswere in size averageof 3.2nanometer, these nanoparticles showed significant antimicrobial and antioxidant activities. It is also reported that Chlorella pyrenoidosa, produceda AgNPs with wide degree of morphologicaluniformity, and it showed a considerable antibacterial activity (Aziz et al., 2015). These biosynthesized AgNPs givingsuch a promising successful approaches in the pharmaceutical industry.
Green Synthesis by plant extracts
The use of plant extracts for the synthesis of AgNPs has tookmuch interest, due to its fast, eco-friendly,nonpathogenic and low costmethodwhich providesa one-stepprocedure for the synthetic nanoparticles processes. Both reduction and stabilization of silver ions (Ag+)are done by active biomolecules present in the plantsfor example; proteins, poly-saccharides, alkaloids, phenolics, saponins, glycosidesand vitamins which are normallypresent in theplant extracts which have medicinal values and are environmentalbenign.Many plants are reported to have a major role in AgNPssynthesiswhich are represented in Table1.
The protocol for the nanoparticle synthesisinclude: the collection of the different parts of plant of interest from their sites and washing thoroughly with tap water to remove both epiphytes and necrotic plants. Then, washing with sterile distilled water to remove associated wasted if any. These, clean and fresh sources are shade-dried for ten days and then powdered using domestic blender. To prepare plant broth, around 10 g of the dried powder is soaked in 100 mL of deionized distilled water (percolation method). The resulting infusion is then filtered thoroughly until no insoluble material appeared in the broth. To 1 mM AgNO3 solution, few milliliters of plant extract were added for the reduction of pure Ag+ ions to Ag˚ which can be further monitored by measuring the UV–visible spectra of the resulting solution at regular intervals(Kulkarni and Muddapur, 2014).
Table1. Green synthesis of silver nanoparticles using different plant extracts.
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Applications of silver nanoparticles:
- The most widely uses of silver nanoparticles are the following:
- Health industry.
- Food storage.
- Textile coating.
- Water treatment.
- Antimicrobial activity of AgNPs:
Theantimicrobial properties of silver nanoparticles have also beenexploited both in the medicine and at daily home use. Silver sulfadiazinecreams use sometimes to prevent infection at the burn site andmany appliance company has incorporated silver intotheir washing machines.Currently, silver is used in the expandingfield of nanotechnology and appears in many consumerproducts that include baby pacifiers, acne creams, and computer’skeyboard, clothing (e.g. socks and athletic wear) that protects from emitting body odor in addition to deodorizingsprays.
Silver nanoparticles synthesized through green methodhave been reported to have biomedical applications as wellas in controlling the pathogenic microbes. In a study, AgNPs were synthesized using aqueous Piper longum fruit extract. The aqueous P. longum fruit extract and the green synthesized silver nanoparticles showed powerful antioxidant properties (Reddy et al., 2014).
- Antiviral activity of AgNPs:
Silvernanoparticles have proven to exert antiviral activity against HIV-1 at non cytotoxic concentrations, but the mechanism underlying their HIV-inhibitory activity has been not fully elucidated. These silver nanoparticles were evaluated to elucidate their mode of antiviral action against HIV-1 using a panel of different in vitro assays (Lara et al., 2010). Special interest has been directed at providing enhanced biomolecular diagnostics, including SNP detection gene expression profiles and biomarker characterization. These strategies have been focused on thedevelopment of nanoscale devices and platforms that can beused for single molecule characterization of nucleic acid,DNA or RNA, and protein at an increased rate when comparedto traditional techniques(Goyal et al., 2009).
- It is a well-known fact that silver nanoparticles and their composites show greater catalytic activities in the area of dye reduction and their removal. Thereduction of methylene blue by arsine in the presence of silver nanoparticle was studied by (Kundu et al., 2002).
- Cytotoxicity of AgNPs:
The toxicity of starch-coated AgNPs was studied using normal human lung fibroblast cells (IMR-90) and human glioblastoma cells (U251). The toxicity was evaluated using changes in cell morphology, cell viability, metabolic activity, and oxidative stress. These nanoparticles produced ATP content of the cell causing damage to mitochondria and increased production of reactive oxygen species (ROS) in a dose-dependent manner. DNA damage, as measured by single cell gel electrophoresis (SCGE) and cytokinesis blocked micronucleus assay (CBMN), was also dose-dependent and more prominent in the cancer cells (AshaRani et al., 2009).
- Safety of AgNPs:
The products prepared with AgNPs have been approved by many accredited agencies and organizations including the US FDA & SIAA of Japan and Research Institute for Chemical Industry (Veeraputhiran, 2013).
Short review on Phyllanthus emblica, Psidium guajava and Lawsoniainermis.
A. Phyllanthus emblica
Phyllanthus emblica L. (syn. Emblica officinalis) is commonly known as Indian gooseberry. InAyurveda, P. emblica has been extensively used, both as edible plants and for its therapeutic potentials. P. emblica is highly nutritious and is reported as an important dietary source of vitamin C, minerals and amino acids.All parts of the plant are used for medicinal purposes, especially the fruit, which has been used in Ayurveda as apotent drug for many health disorders.
The local name of P. emblica L. in Nepal is Amala. The other common names of this plant include, Ganlanshu, Youganzi (Chinese); Emblic myrobalan, Indian Goose berry (English); Kemloko (Japanese); Chu me, Kam lam (Vietnames). Ciccaemblica, Emblica officinalis, Mirobalanusembilica, P. mairei, P. laxifolius, Dichelastinanodicaulis are the synonyms of P. emblica.
It can be classified as kingdom: Plantae; division: flowering plant; class: Magnoliopsida; order: Malpighiales; family: Euforbiaceae /Phyllanthaceae; tribe: Phyllantheae; subtribe: Flueggeinae; genus: Phyllanthus; species: P. emblica.
Phyllanthusemblica L. is a tree of small or moderate size with a greenish-grey bark and greenish-yellow flowers, formed in axillary clusters. Branchlets are alternate superposed and they all face in one plane. Length of the branchlets is up to 40 cm and more than 100 leaves are arranged in the branchlets. Barks are brown in color and peels into small irregular flake. The miniature, oblong leaves, only 3 mm wide and1.25–2 cm long, distichously disposed on branchlets,give a misleading impression of finely pinnate foliage.The feathery leaves are linear-oblong, with a roundedbase and obtuse or acute apex. It has axillary cymes,densely fascicled along the leaf bearing branchlets,often on the naked portion below the leaves. The arms from thetip of the ovary measure about 5–7 mm long. The fruitdiameter ranges between 1.8 and 2.5 cm. Groovemarkings along the septa are very shallow. The tenderfruits are green, fleshy, globose and shining, andchange to light yellow or brick-red when mature(Arora et al., 2012).
Ascorbic acid (vitamin C) is the most plentiful constituents of P. emblica fruit. Beside it, otherphytochemicals compounds isolated from this plant includefixed oils, phosphatides, essential oils, tannins,minerals, vitamins, amino acids, fatty acids and glycosides (Bhattacharya et al., 1999).
Fatty acids reported from from P. emblica include, linolenic, linoleic, oleic, stearic, palmitic and myristic acids. Besides, D-glucose, D-fructose, D-myo-inositol, D-galacturonic acid, D-arabinosyI, D-rhamnosyl, D-xylosyI, D-glucosyI, D-mannosylandD-galactosyI residues are the sugars.
Emblicanin Aand Emblicanin B, pedunculaginand punigluconinare the major tannins reported from this plant(Figure3&4).
Abbildung in dieser Leseprobe nicht enthaltenAbbildung in dieser Leseprobe nicht enthalten Figure3. Chemical structure of Emblicanin A & B tannins, (Author’s own work).
Abbildung in dieser Leseprobe nicht enthalten
Figure4. Chemical structure of pedunculagin & punigluconin, (Author’s own work)
Other compounds isolated from this plants aregallic acids, amlaic acid, arginine, aspartic acid,astragallin, Beta carotene, β-sitosterol, chebulagicacid, chebulic acid, chebulaginic acid, chebulinicacid, corilagic acid, corilagin, cysteine, ellagicacid, gibberellins, glutamic acid, glycine,histidine, isoleucine, kaempferol, leucodelphinidin,methionine, phenylalanine, phyllantidine, phyllemblin (Figure 6)quercetin, riboflavin, rutin, thiamin, threonine,tryptophan, tyrosine, valineand zeatin(Figure5)(Gaire and Subedi, 2014).
Abbildung in dieser Leseprobe nicht enthalten
Figure5. Chemical structures of selected compounds from P. emblica, (Author’s own work)
Abbildung in dieser Leseprobe nicht enthalten
Figure 6. Chemical structures ofamlaic acid,phyllemblin andphyllantidine, (Author’s own work).
Pharmacological and Therapeutic Activity :
Antioxidantactivity: Previously, many researches have been studiedthe antioxidant and free radical scavengingactivity of P. emblica and the main reason behindthis is ascorbic acid, tannins and polyphenolicconstituents of P. emblica. Different extract of P.emblica and phytoconstituents are reported to haveantioxidant activity against several free radicals suchas 1,1-diphenyl-2-picrylhydrazyl (DPPH) freeradicals, superoxide and nitric oxide. Phytochemicalsfrom P. emblica are also reported as good metal ionchelator as it can prevent the oxidative cascades(Poltanov et al., 2009).
Antimicrobial effect:Aqueous extract of P. emblica showedpotent antimicrobial activity against Enterobacter cloacae and Klebsiella pneumoniae. Aqueous infusion of E. officinalis exhibited high antibacterial activity against E. coli, K. ozaenae, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi, S. paratyphi A, S. paratyphi B and Serratia marcescens. Phyllanthus emblica leaf extract wasreported to possess antimalarial potency with the50% inhibitory concentration (IC50) 7.25 µg/mL forethyl acetate extract and 3.125 µg/mL for methanolextract against Plasmodium falciparum parasite(Bagavan et al., 2011).
Anti-inflammatory activity: Phyllanthus emblica reported to possess a powerful anti-inflammatoryactivity against compound 48/80-induced paw edemain both Balb/c mice and Swiss Albino mice, also againstcarrageenan-induced paw edema in Wistar albino rats. It exhibited a dose-dependent anti-inflammatory effect against Freund's adjuvant-induced arthritis in Wistar Albino rats and an approximately63% inhibitory effects and good trypsin inhibitoryactivity. Furthermore, the water fraction of P. emblica fruits from butanol extract has powerful potential anti-inflammatory activity against indomethacin-induced gastric ulcer(Bandyopadhyay et al., 2000).
Laxative effect: The fresh ripe fruits are used extensively in Indiaas a laxative, and one or two fruits being sufficient fora single dose. They have been exported to Europe,preserved in sugar, and are valued as a pleasantlaxative for children and made into a confectionconsisting of the pulp of the de-seeded fruit. It alsopossesses prokinetic and laxative activities in micealong with spasmodic effect in the isolated tissuesof guinea pig and rabbit, and this action is mediatedpartially through activation of muscarinic receptors,suggesting a rationale for the medicinal use of P. emblica fruits in indigestion and constipation(Mehmood et al., 2013).
Antidiarrheal effect: In traditional medicine, fruit decoction of P. emblica is mixed with milk and given in cases ofdysentery. Infusion of the E. officinalis leaves with fenugreek seedsare given for chronic diarrhea. Recent report suggest thatthe P. emblica fruit extract possesses antidiarrheal andspasmolytic activities, which may be mediated possibly through dualblockade of muscarinic receptors and Ca2+ channels, which explaining its medicinal use in diarrhea(Mehmood et al., 2013).
Hepatoprotective effect: Phyllanthus emblica and its flavonoid quercetin were foundto be hepatoprotective against acetaminopheninducedliver damage in Albino rats as long as mice. The obtainedresults indicated that the possible mechanism of P. emblica for hepatoprotective activity is in reducingglutathione depletion and inhibit the stimulation ofcytochrome P450. Since, quercetin flavonoid alone was moreeffective than the P. emblica extract, it is thought tobe the active material in the extract. Toxic effects induced by leadnitrate and aluminum sulphate were also diminishedby the administration of P. emblica extract and ascorbicacid in albino rats. It is also reported that E. officinalis has strong hepatoprotective effect against hepatic damage induced by carbontetrachloride (Lee et al., 2006).
Cardioprotective activity: Bioactive tannoid principles of P. emblica were investigatedfor its antioxidant effect which was carried out on cardiac ischemic-reperfusion-induced oxidative stress in the heart of rats. In this study, fraction enriched of emblicanin A and emblicanin B fresh juice of P. emblica fruits was extracted with aqueous methanol fraction and used for the experiment. The extract of P. emblica and vitamin E (as standard oxidizing agent)were administered orally twice daily for 2 consecutive weeks prior to the perfusion experiments. The resultsdemonstrated that P. emblica significantly reversedthe effects of ischemia-reperfusion on major antioxidantenzymes such as superoxide dismutase, catalase, glutathione peroxidase and lipid peroxidation activities. The claim that antioxidants of P. emblica may act as cardioprotective agents is supported with this study(Bhattacharya et al., 2002).
Anticancer activity: Aqueous extract of P. emblica exhibited anticancer activity against mouse skin tumerogenesisby decreasing the tumor number and volume and itwas found to be very effective against breast cancer.As well as, aqueous extract of fresh P. emblica fruits at a concentration of 16.5 mg/mL was found tosignificantly reduce the growth of cytotoxic L929 cellsin culture. Extract of P. emblica was also found tosignificantly reduce solid tumors induced by Daltonlymphoma ascites cells whereas having only amoderate effect on ascites tumor(Jose et al., 2001).
Immunomodulatory effect: Research experiment in mice exhibited that the aqueous extract of P. emblica is a natural killer cell activityand antibody-dependent cellular cytotoxicity in mice.Ethanol extract of P. emblica also exhibited biphasic activityin ulcerated mice with dose-dependent healing effect.It significantly reduced pro-inflammatory cytokine,tumor necrosis factor α (TNF-α) and interleukin-1β(IL-1β) levels and up-regulate the anti-inflammatory cytokine (IL-10) concentration(Chatterjee et al., 2011).
Antitussive effect: The antitussive activity of P. emblica was examined in conscious cats by mechanical stimulation ofthe laryngopharyngeal and tracheobronchial mucousareas of airways. The fruits ethanol extract of P. emblica seems to have a good ability to reduce the mechanically-provoked cough, but only at higher doses(200 mg/kg body weight) suggesting the presence of significant antitussive activity of P. emblica in conscious cats. Which is dose-dependent but higher than the antitussiveactivity of the much used non-narcotic antitussivedrug, dropropizine. It is supposed that the antitussiveactivity of the extract of P. emblica is due to, notonly antiphlogistic, antispasmolytic and antioxidant activity effects, but also to its effect on mucussecretion in the airways of the cats (Nosal’ova et al., 2003).
B. Psidium guajava
Psidium guajava Linn. (Myrtaceae), is an important food and medicinal plant in tropical and subtropical countries, which is widely used as food and in folk medicine around the world. This is due to comprehensive chemical constituents, pharmacological, and clinical uses. Many pharmacological experiments have been carried out in a number of in vitro and in vivo models. Also,phyto-constituents have been identified to which many of the medicinal importances are attributed. A number of secondary metabolites are present in P. guajava in a good yield and some have been shown to significantly possess useful biological activities.These secondary metabolites are mainly belonging to phenolics, flavonoids, carotenoids, terpenoids and triterpenes. Taxonomy:
It can be classified as kingdom: Plantae; order: Myrtales; family: Myrtaceae; genus: Psidium; species: P. guajava.
Morphology & habitat :
It is a low evergreen tree or shrub 6 to 25 feet high, contains wide-spreading branches and square, downy twigs, is a native of Middle East, Africa, tropical America and Asia. It also has common names as, common guava, yellow guava and apple guava.It is a common vegetation cover roads and in waste places in Hawaii. Psidium guajava is a tropical and semitropical plant. It is well known for its edible fruit. It is common in the backyards and gardens. The branches are crooked, bringing opposite leaves. The flowers are white, incurved petals, 2 or 3 in the leaf axils, usually they are fragrant, with four to six petals and yellow anthers. The fruit known for its small size, 3 to 6 cm long, pear-shaped, reddish-yellow when ripe.Fruit are fleshy yellow globose to ovoid berry about 5 cm in diameter with an edible mesocarp containing numerous small hard white seeds. Psidium guajava leaves are opposite, short-petiolate, the blade oval with prominent pinnate veins, 5 to 15 cm long(Gutiérrez et al., 2008).
The fruits are characterized by a low content of carbohydrates(13.2%), fats (0.53%), and proteins (0.88%) and by a highwater content (84.9%). Food value per 100 g is: Calories 36 to 50 kcal, moisture 77 to 86 g, crude fiber 2.8 to 5.5 g, ash 0.43 to 0.7 g, calcium 9.1 to 17 mg, phosphorus 17.8 to 30 mg, iron 0.30 to 0.70 mg, vitamin A 200–400 I.U., thiamine 0.046 mg, riboflavin0.03 to 0.04 mg, niacin 0.6 to 1.068 mg, ascorbic acid 100 mg, vitaminB3 40 I.U.Manganese is also present in the plant in combinationwith phosphoric, oxalic and malic acids. 3-caryophyllene (24.1%), nerolidol (17.3%), 3-phenylpropylacetate (5.3%) and caryophyllene oxide (5.1%) were isolatedfrom essential oil extracted from the fruits. Thereafter, the active aromatic constituents present in guava fruit the 3-penten-2-ol and 2-butenyl acetate were isolated. The fruit also contains glykosen 4.14%,saccharose 1.62%, and protein 0.3%(Gutiérrez et al., 2008).
The unripe fruit is indigestible, causes vomiting and feverishness.It changes in chemical composition and the activities ofhydrolytic enzymes (the activities of α-amylase and β-amylasereduced significantly with ripening), chlorophyll, cellulose,hemicellulose, and lignin content increased while carotenoidcontent decreased. Tannins are present in high concentration in the unripe fruit, which is astringentand has a tendency to cause constipation, but it is sometimesemployed in diarrhea(Jain et al., 2003).
Leaves of Psidium guajava contain essential oil with the main componentsbeing α-pinene, β-pinene, limonene, menthol, terpenyl acetate,isopropyl alcohol, longicyclene, caryophyllene, β-bisabolene,cineol, caryophyllene oxide, β-copanene, farnesene, humulene,selinene, cardinene and curcumene(Begum et al., 2004).
Additionally, flavonoids, and saponins combined witholeanolic acid have been isolated from the P.guajava leaves. Nerolidiol, β-sitosterol, ursolic, andguayavolic acids have also been identified. In addition,the leaves contain triterpenic acids as well asfixed oil 6%, 3.15% resin, and8.5% tannin, and a number of other fixed substances such as, fat, cellulose,tannin, chlorophyll and mineral salts(Gutiérrez et al., 2008).
Also, guavanoic acid, guavacoumaric acid, 2α-hydroxyursolic acid, jacoumaric acid, isoneriucoumaric acid, asiatic acid, and β-sitosterol-3- O -β-glucopyranoside have been isolated from the leaves of P. guajava (Begum et al., 2002).
On the other hand, in mature leaves, the greatest concentrations of flavonoids were found in July: myricetin (208.44 mg kg−1), luteolin (51.22 mg kg−1), quercetin (2883.08 mg kg−1) and kaempferol (97.25 mg kg−1) (Figure7). Two triterpenoids, 20β-acetoxy-2α,3β-dihydroxyurs-12-en-28-oic acid which is known as (guavanoic acid), and 2α,3β-dihydroxy-24- p - z -coumaroyloxyurs-12-en-28-oic acid which is known as (guavacoumaric acid), along with six known compounds 2α-hydroxyursolic acid, jacoumaric acid, isoneriucoumaricacid, asiatic acid, ilelatifol d and β-sitosterol-3- O -β-glucopyranoside,have been isolated from the leaves of P.guajava. guajavolideand guavenoic acid, were also isolated fromfresh leaves of P.guajava (Gutiérrez et al., 2008).