Research Articles

2019  |  Vol: 5(2)  |  Issue: 2(March-April)  |  https://doi.org/10.31024/ajpp.2019.5.2.17
Green synthesis and characterization of gold nanoparticles using Bursera serrata fruit extract

Kausik Chaudhuri1, 2, Sk Nurul Hasan1, Abir Chandan Barai1, Subhajit Das1, Tapan Seal2*, Braja Gopal Bag1*

1Department of Chemistry and Chemical Technology, Vidyasagar University, Midnapore 721102,  West  Bengal, India

2Plant Chemistry Department, Botanical Survey of India, Acharya Jagadish Chandra Bose Indian Botanic Garden, Shibpur, Howrah-711103, West Bengal, India

*Address for Corresponding Author

Braja Gopal Bag

Department of Chemistry and Chemical Technology, Vidyasagar University, Midnapore 721102, West Bengal, India


Abstr​act

Objective: Nano-chemistry is an emerging field with its presence felt in every sphere of life. The synthesis of nanoparticles using plant extract is alternative to the conventional methods with deleterious effects on the environment. Materials and methods: The different antioxidants present in plant extract act as reducing and stabilising agent for the synthesis of Bursera serrata conjugated gold nanoparticles (BS-AuNPs). The fruits of B. serrata were well-characterized for its nutritive potential, mineral content and antioxidant properties in different solvent extracts. The quantitation’s of polyphenolics in the fruits were carried out by High Performance Liquid Chromatography (HPLC) method which indicated the presence of phenolics and polyphenolics in different amounts.  The phytochemicals present in the fruits extract   were utilized for the one-step green synthesis method of B. serrata conjugated AuNPs (BS-AuNPs). Results: The formation of BS-AuNPs was confirmed by Surface Plasmon Resonance spectroscopy (SPR), high resolution transmission electron microscopy (HRTEM), and X-ray diffraction (XRD) analyses. Conclusion: The BS-AuNPs obtained by these biogenic syntheses have potential biological and medical applications depending on their size and aqueous stability.

Keywords: Green synthesis, Bursera serrata, antioxidant, polyphenolics, HPLC, gold nanoparticles


Introduction

Bursera serrata Wall. (Ex Colebr.) belongs to the family Burceraceae, is a large tree generally predominant on foot hills of lower Assam, India. It is considered as very hard wood generally not cut for firewood etc and the fruits are drupe and furrowed. The fruits are edible and yields aromatic oil. The plant showed anti-inflammatory, anti-tumor, agglutinating and immobilizing activities. The stem bark of the plant was found to contain   β-amyrin, β-sitostenone, coumarin, scopoletin (Sayeed et al., 2014).  Nano chemistry opens a new vista in the field of biomedical devices and biotechnology. During the last few decades, metal nanoparticles have elicited much interest due to their distinct physical, chemical and biological properties (Safari and Zarnegar, 2014; Alkilany et al., 2013). In recent years, plant-mediated bio-synthesis of nanoparticles is gaining importance due to its simplicity and eco-friendliness. Such bio-synthesis provides advancement over other methods as it is simple, one step, cost-effective, environment friendly and easily reproducible (Dauthal and Mukhopadhyay, 2016; Kunoh et al., 2017). Metallic nanoparticles exhibit various size and shape-dependent optical properties, which are useful in various biomedical applications like the imaging of specific target cells and tissues, drug delivery, bio-sensing and catalysts to optics (Verma et al., 2016; Ahmad and Sardar, 2015). The remarkable antimicrobial effect of metallic nanoparticles is of interest due to the growing microbial resistance against the antibiotics and development of resistant strains (Ahmed et al., 2016; Sahayaraj and Rajesh, 2011). Among different types of nanomaterials, noble metal nanoparticles gained considerable attention due to their special catalytic, electronic, and optical properties. Nanoparticles are of immense interest due to their extremely mini-scale size and high surface area to volume ratio, which lead to both chemical and physical differences in their properties compared to the bulk having the same composition (Rodriguez et al., 2014). The interest in gold nanoparticles (AuNPs) is largely due to the relative ease of their synthesis, with good control of their sizes and shapes, their optical characteristics and their good bio-compatibility. Plant-based synthesis of AuNPs, via the reduction of Au(III), is relatively faster and safer and, does not require any additional stabilizer.  Moreover, the synthesis can be carried out at room temperature without the need of high physical and instrumental requirements under easy to handle and easy to scale-up and reaction procedures. Gold nanoparticles have been widely investigated due to their uniqueness especially in bio-medication and in bio-imaging. Moreover, the mixture of AuNPs and green reductants may possibly result in synergistic biological activities. Various plant parts (roots, stems, bark, leaves and petals) can be exploited as reducing and stabilizing agents in the green synthesis of AuNPs (Awwad et al., 2013; Babu et al., 2011).

The present study was conducted to evaluate the nutraceutical properties, antioxidant activities, quantitation of polyphenolics by HPLC and one step bio-synthesis of gold nanoparticles using the fruits extract of B. serrata.  Moreover, the phytochemicals present in the fruits extract of B. serrata were utilized for the green synthesis of B. serrata conjugated AuNPs (BS-AuNPs) at room temperature in water under very mild reaction condition.  The synthesized BS-AuNPs were characterized by Surface Plasmon Resonance spectroscopy, high resolution transmission electron microscopy (HRTEM) and X-ray diffraction (XRD) analyses.

Materials and methods

Plant materials

The fruits of B. serrata were collected from Meghalaya state, India in December, 2015 and identification was authenticated in our office of Shibpur Botanical garden, Shibpur, India. The voucher specimens were preserved in the Plant Chemistry department of our office under registry no BSITS 109. The plant parts were shed-dried, pulverized and stored in an airtight container for further extraction.

Chemicals 

The standard phenolic acids (gallic acid, protocatechuic acid, gentisic acid, chlorogenic acid, p-hydroxy benzoic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid, salicylic acid and ellagic acid), flavonoids (catechin, rutin, myricetin, quercetin, naringin, apigenin and kaempferol),1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), butylated hydroxytoluene (BHT) were procured from Sigma Chemical Co. (St. Louis, MO, USA). Folin-Ciocalteus’s phenol reagent, potassium ferricyanide, potassium per sulphate, aluminium chloride, ferric chloride, anthrone, sodium carbonate,  HPLC-grade solvents (acetonitrile, methanol, water and trifluoroacetic acid), sodium dihydrogen phosphate were purchased from Merck (Germany). Tetrachloroauric acid (HAuCl4) was purchased from SRL. All the chemicals and solvents used were of analytical grade.

HPLC equipment 

Dionex Ultimate 3000 liquid chromatograph   attached with a diode array detector (DAD) was taken for HPLC analysis. The separation of components was achieved by a reversed-phase Acclaim C18 column (5 micron particle size, 250 x 4.6 mm). 20 𝜇L of sample was injected into the HPLC column. The Chromeleon system manager was used for analyzing the data.

Proximate composition and minerals content in B. serrata

Estimation of ash content

Five grams of powdered fruits of the plant were taken in a silica crucible and heated for about 5-6 h in a muffle furnace controlled at 500°C.The crucible was cooled, weighed and heated again in the furnace for half an hour. This process was repeated consequently until the weight of the crucible along with sample became constant (ash became white or greyish white). Weight of ash gave the ash content (Seal et al., 2017).  The ash obtained was preserved for mineral analysis.

Ash content (%) = Weight of ash × 100/Weight of sample

Estimation of moisture content

The moisture content of the plant sample was carried out by heating a known amount of fresh fruits in an air oven at 100-110°C and weighed. The loss in weight was considered as a measure of moisture content in the sample (Seal et al., 2017).

Moisture (%) = [(Weight of original sample−Weight of dried sample)] x100 /Weight of original sample

Estimation of crude fat content

Two gm of moisture free fruits were soxhleted with petroleum ether (40-60°C) for about 6-8 h. The petroleum ether extract was filtered and evaporated in a pre-weighed beaker. Increase in weight of a beaker determines crude fat content. Percentage of fat content was calculated using the following formula (Seal et al., 2017).

Crude fat (%) = Weight of fat in sample × 100/Weight of dry sample

Estimation of crude fibre content

The crude fibre content in the plant sample was carried out by warming two gm of moisture and fat-free fruits with 200 ml of 1.25% sulphuric acid followed by 1.25% sodium hydroxide solution and with 1% nitric acid. The solution was filtered and the residue was washed with boiling water and then the residue was dried in an oven at 130°C to constant weight. The residue was heated in muffle furnace at 550°C for two hours, cooled in desiccators and weighed. The crude fibre content was expressed as percentage loss in weight on ignition (Seal et al., 2017).  

Crude fibre (%) = (Weight of residue − Weight of ash) x 100/ Weight of the sample

Estimation of crude protein content

The micro Kjeldahl method was adopted for the estimation of crude protein content in the plant where two gm of samples were digested with concentrated sulphuric acid in a Kjeldahl flask in the presence of 0.5 gm CuSO4 and 5 gm K2SO4, until a clear solution was obtained. The digested solution was cooled and diluted with distilled water and an excess of sodium hydroxide solution (40%) was added to the diluted reaction mixture, the liberated ammonia was distilled in steam and absorbed in 25 ml (N/20) sulphuric acid. The excess mineral acid was titrated with known strength of sodium hydroxide and from this the percentage of nitrogen in the sample was calculated. The amount of protein content was determined by multiplying the amount of nitrogen with 6.25 (Seal et al., 2017).

Estimation of carbohydrate content 

100 mg of fruits were hydrolysed with 5 ml of hydrochloric acid (2.5 N), cooled to room temperature and neutralised with solid sodium carbonate until the effervescence ceases. The solution filtered in a 100 ml volumetric flask and make up the volume with distilled water. To 1 mL of this solution, 4 ml freshly prepared anthrone reagent (200 mg anthrone dissolved in 100 ml of ice-cold 95% sulphuric acid) were added and heated in a water bath for eight minutes. The mixture was cooled rapidly, a dark green colour appeared and the absorption at 630 nm was measured (UV-visible spectrophotometer Shimadzu UV 1800). The total carbohydrate content was expressed as glucose equivalents   using the following equation based on the calibration curve y = 0.0081x + 0.2475, R2 = 0.9993 where y was the absorbance and x concentration of glucose in mg/ml (Hedge and Hofreiter, 1962).

Estimation of energy content

The energy (kcal/100gm) content of plant sample was determined by multiplying the values obtained for protein, fat and available carbohydrate by 4.00, 9.00 and 4.00 respectively and adding up the values (Guil-Guerrero et al., 1998).

Estimation of minerals

One gram of ash of the plant obtained above was dissolved in 30 ml of hydrochloric acid (5 % ) solution, filtered and volume make up to 50 ml with double distilled water and minerals were estimated in atomic absorption spectrophotometer (AAS) (AA 800, Perkin-Elmer Germany). The standard solution of each element was prepared and calibration curves were drawn for each element using AAS (Indrayan et al., 2005). All assays were carried out in triplicate and values were obtained by calculating the average of three experiments and data are presented as Mean ± SEM.

Antioxidant activities of B. serrata

Extraction of plant material (Benzene, chloroform, acetone and methanol)  

One gram of each plant materials were extracted with 20 ml each of benzene, chloroform, acetone and methanol with agitation for 18-24 h at ambient temperature. The extracts were filtered and diluted to 50 ml and aliquot were analyzed for their total phenolic, flavonoid and flavonol content, reducing power and their free radical scavenging capacity.

Estimation of total phenolic content 

The amount of total phenolic content of crude extracts was determined according to Folin-Ciocalteu procedure (Seal and Chaudhuri 2015). The tested samples (20 - 100 ml) were taken into test tubes. 1 ml of Folin-Ciocalteu reagent and 0.8 ml of sodium carbonate (7.5%) were added. The contents were mixed and allowed to stand for 30 min. Absorption at 765 nm was measured (UV-visible spectrophotometer Shimadzu UV 1800). The total phenolic content was expressed as gallic acid equivalents (GAE) in mili gram per gram (mg/g) of extract using the following equation based on the calibration curve  y = 0.0013x + 0.0498,  R2 = 0.999 where y was the absorbance and x was the Gallic acid equivalent (mg/g).

Estimation of total flavonoids

Total flavonoids were estimated using the method described at Seal and Chaudhuri 2015. To 0.5 ml of sample, 0.5 ml of 2% AlCl3 in ethanol was added. After one hour, at room temperature, the absorbance was measured at 420 nm (UV-visible spectrophotometer Shimadzu UV 1800). A yellow color indicated the presence of flavonoids. Total flavonoid contents were calculated as rutin equivalent (mg/g) using the following equation based on the calibration curve: y = 0.0182x - 0.0222, R2 = 0.9962, where y was the absorbance and x was the Rutin equivalent (mg/g).

Estimation of total flavonols

Total flavonols in the plant extracts were estimated using the method stated at Seal et al., 2017. To 2.0 ml of sample (standard), 2.0 ml of 2% AlCl3 ethanol and 3.0 ml (50 g/L) sodium acetate solutions were added. The absorption at 440 nm (UV-visible spectrophotometer Shimadzu UV 1800) was read after 2.5 h at 20°C. Total flavonol content was calculated as quercetin equivalent (mg/g) using the following equation based on the calibration curve:  y = 0.0049x + 0.0047, R2 = 0.9935, where y was the absorbance and x was the quercetin equivalent (mg/g).

Measurement of reducing power 

The reducing power of the extracts was determined according to the method described by Seal et al. (2017). Extracts (100 µl) of plant extracts were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and 1% potassium ferricyanide (2.5 ml). The mixture was incubated at 50°C for 20 min. Aliquots of 10% trichloroacetic acid (2.5 ml) were added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and a freshly prepared ferric chloride solution (0.5 ml, 0.1%). The absorbance was measured at 700 nm. Reducing power is given in ascorbic acid equivalent (AAE) in milligram per gram (mg/g) of dry material using the following equation based on the calibration curve: y = 0.0023x - 0.0063, R2 = 0.9955 where y was the absorbance and x was the ascorbic acid equivalent (mg/g).

Determination of DPPH free radical scavenging activity

The free radical scavenging activity of the plant samples and butylated hydroxyl toluene (BHT) as positive control was determined using the stable radical DPPH (1,1-diphenyl-2-picrylhydrazyl) (Seal et al., 2017). Aliquots (20 -100 ml) of the tested sample were placed in test tubes and 3.9 mL of freshly prepared DPPH solution (25 mg L-1) in methanol was added in each test tube and mixed. The absorbance was measured at 517 nm (UV-visible spectrophotometer Shimadzu UV 1800) after 30 min. The capability to scavenge the DPPH radical was calculated, using the following equation:

DPPH scavenged (%) = {(Ac – At)/Ac} x 100

Where Ac is the absorbance of the control reaction and At is the absorbance in presence of the sample of the extracts. The antioxidant activity of the extract was expressed as IC50. The IC50 value was defined as the concentration in mg of dry material per ml (mg / ml) that inhibits the formation of DPPH radicals by 50%. Each value was determined from regression equation.

Scavenging activity of ABTS radical cation

The 2,2´-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation (ABTS.+)-scavenging activity was measured according to the method described by (Seal et al., 2017). ABTS was dissolved in water to a 7 mM concentration. The ABTS radicals were produced by adding 2.45 mM potassium persulphate (final concentration).The completion of radical generation was obtained in the dark at room temperature for 12–16 h. This solution was then diluted with ethanol to adjust its absorbance at 734 nm to 0.70 ± 0.02. To determine the scavenging activity, 1 mL of diluted ABTS.+ solution was added to 100 ml of plant extract (or water for the control), and the absorbance at 734 nm was measured 6 min after the initial mixing, using ethanol as the blank. The percentage of inhibition was calculated by the equation:

ABTS scavenged (%) = (Acont - Atest) / Acont´ 100

Where, Ac and As are the absorbencies of the control and of the test sample, respectively. From a plot of concentration against % inhibition, a linear regression analysis was performed to determine the IC50 value of the sample.

Quantification of phenolic acids and flavonoids in the methanol extract of   B. serrata by HPLC

Preparation of standard solutions 

The stock solution of gallic acid of concentration 1 mg/ml was prepared by dissolving 10 mg gallic acid in  1 mL HPLC-grade methanol followed by sonication for 10 min  and the resulting volume was made up to 10 mL with the solvent for the Mobile phase (methanol and 0.5% aq. acetic acid 1:9).   The same method was followed to prepare the standard stock solutions of the phenolic acids and the flavonoids viz. protocatechuic acid, gentisic acid, chlorogenic acid, p-hydroxy benzoic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid, salicylic acid and ellagic acid, catechin, rutin, myricetin, quercetin, naringin, apigenin and kaempferol. The working standard solutions of concentrations 20, 40, 60, 80 and 100μg/ml were prepared by further dilution of the standard solution with the mobile phase solvent system.  The standard and working solutions were filtered through 0.45 μm PVDF-syringe filter and the mobile phase was degassed before the injection of the solutions.

Chromatography analysis for quantification of phenolic acids and flavonoids

HPLC analyses for the quantification of phenolic acids and flavonoids in the plant extract were performed following the method described by (Seal et al., 2017) with minor modification. The analysis were carried out using Dionex Ultimate 3000 liquid chromatograph  including a  diode array detector (DAD) with 5 cm flow cell and with Chromeleon system manager as data processor. The separation was achieved by a reversed phase Acclaim C18 column (5 micron particle size, 250 x 4.6 mm). 20 𝜇L of sample was introduced into the HPLC column. The method was validated according to the USP and ICH guidelines (ICH-Q2A 1995, ICH-Q2B 1996). The mobile phase contains methanol (Solvent A) and 0.5% aq. acetic acid solution (Solvent B) and  the column was thermostatically controlled at 25 0C and the injection volume was kept at 20 μl.  A gradient elution was performed by varying the proportion of solvent A to solvent B.  The gradient elusion was  10 % A and 90% B with flow rate 1 ml/min to 0.7 ml/min in 27 min, from 10 to 40 % A with flow rate 0.7 ml/min for 23 min, 40% A and 60% B with flow rate 0.7 ml/min initially for 2 min and then  flow rate changed from 0.7 to 0.3 ml/min in 65min, from 40 to 44% A with flow rate 0.3 to 0.7ml/min in 70 min, 44% A with flow rate 0.7 to 1ml/min for 10 min duration, solvent A changed from 44%  to 58 %  with flow rate 1ml/min for 5 min, 58 to 70% A in 98 min at constant flow rate 1 ml/min.  The mobile phase composition back to initial condition (solvent A: solvent B: 10: 90) in 101 min and allowed to run for another 4 min, before the injection of another sample. Total analysis time per sample was 105 min.

HPLC chromatograms were detected using a photo diode array UV detector at three different wavelengths (272, 280 and 310 nm) according to absorption maxima of analysed compounds. Each compound was identified by its retention time and by spiking with standards under the same conditions.  The quantification of phenolic acids and flavonoids in the fruits of the plant were carried out by the measurement of the integrated peak area and the contents were calculated using the calibration curve by plotting peak area against concentration of the respective standard sample.

Biosynthesis of gold nanoparticles using methanol extract of B. serrata

Preparation of Au (III) solution

HAuCl4 was purchased from SRL (Sisco Research Laboratory) and used without further purification. HAuCl4 (52.8 mg) was dissolved in deionized water (10 mL) to obtain a 13.4 mM Au(III) stock solution.

Preparation of the fruit extract of B. serrata

Finely powdered fruits of B. serrata (3 gm) was suspended in methanol (10 ml) in a test tube, sonicated in an ultrasonicator bath for 45 min and then centrifuged for 10 minutes to obtain a clear supernatant.  To know the concentration of the fruit extract, an aliquot of the clear supernatant (2 ml) was taken in a round bottom flask and the volatiles were removed under reduced pressure to afford a sticky solid (1.7 mg).  Thus the concentration of the fruit extract was 1700 mg/ l (Hasan et al., 2017).

Synthesis of Gold Nanoparticles

Aliquots of Au (III) solution (0.2 ml, 13.4 mM each) were added drop-wise to the solution of fruits extract of B. serrata to prepare a series of stabilized AuNPs where concentration of the extract were 100, 200, 300 and 400 mgL-1 and the concentration of Au (III) was fixed at 0.67 mM. UV-visible spectroscopy of the solutions was carried out after 24 h of HAuCl4 and the leaf extract of the plant had been mixed (Hasan et al., 2017).

Characterization

HRTEM images, SAED and EDX of AuNPs were taken from Technai G2 instrument. UV-visible spectra were recorded in Shimadzu 1601 spectrophotometer. X-ray diffraction (XRD) patterns of the stabilized AuNPs were recorded Bruker-D8 Advanced with Cu-Kα radiation (λ= 1.54 Ǻ).  

Results and discussion

Proximate composition and minerals content in B .serrata

The fruits of B. Serrata were taken for the analysis of proximate composition. The proximate composition of the plant is appended in table 1.

The proximate analysis of the plant showed that 100 gm of dry plant contain 10.24 ± 0.211 gm ash and 85.32 ±0.23 gm moisture. The high amount of ash content indicated that this plant was rich in minerals and could provide a substantial amount of mineral elements to our diet (Satter et al., 2016).

Table 1. Proximate composition and minerals content in B. serrata

Proximate composition

Amount

Minerals

Amount (mg/g)

Ash (%)

10.24 ± 0.211

Sodium (Na)

0.94 ± 0.08

Moisture (%)

85.32 ± 0.23

Potassium  (K)

10.74 ± 0.32

Protein (%)

2.10 ± 0.02

Calcium (Ca)

14.43 ± 0.30

Fat (%)

1.81 ± 0.035

Copper (Cu)

BDL

Carbohydrate (%)

38.64 ± 0.21

Zinc (Zn)

0.070 ± 0.0002

Crude fibre (%)

17.35 ± 0.19

Magnesium  (Mg)

0.757 ± 0.003

Energy (kcal/100gm)

76.13 ± 0.50

Iron (Fe)

0.018 ± 0.001

-

-

Manganese (Mn)

BDL

Each value in the table was obtained by calculating the average of three experiments and data are presented as Mean ± SEM

The plant was found to contain protein, fat, fibre and carbohydrate 2.1±0.02, 1.81 ±0.035, 17.35 ±0.19 and 38.64± 0.21 respectively. The energy content of the plant was calculated at 76.13±0.5 kcal/100 gm. The fat and fibre content in the plant was particularly high and well compared to that reported for some common vegetables which indicates that the consumption of the plant would be helpful for the absorption of fat soluble vitamins like vitamin A and carotene in the body and might play an important role in decreasing the risks of many disorders such as constipation, diabetes, serum cholesterol, heart diseases, breast and colon cancer, hypertension, etc. (Sundriyal and  Sundriyal, 2004; Koca et al.,2015). The plants are rich sources of protein which can encourage their use in human diets and would be helpful for the proper functioning of antibodies resisting infection (Sundriyal and Sundriyal, 2004).

Fruits, and vegetables, are important sources of macro-minerals (Na, K, Ca, Mg) and micro-minerals (Fe, Zn, Cu, Mn, Pb, Cr) which are responsible for maintaining physiological and biological functions of the human body. The fruits of the plant contain a very good amount of sodium (0.94± 0.08 mg/gm) and potassium (10.74 ± 0.32 mg/gm). The ratio of K/Na was significant in this plant (11.43) which is very much responsible to control the high blood pressure of our body (Sapui et al., 2009). The fruits of the plant was found to contain 14.43 ±0.3 mg/g calcium which might be beneficial to build strong and healthy bones and also required for the normal functioning of the cardiac muscles (Seal and Chaudhuri, 2015).  A sufficient amount of Cu, Zn, Mg, Fe and Zn were present in the plant indicating that the consumption of this vegetable might be helpful for preventing iron- deficiency anaemia, nucleic acid metabolism, control the blood- glucose levels and support a healthy immune system (Ihedioha and Okoye, 2011; Saikia and Deka 2013) .

Antioxidant activities of the different solvent extracts of B. serrata

Extractive value 

The extractive values of the plant under investigation with four different solvents are shown in     table 2.  The results indicate that,  methanol is the most suitable solvent to obtain the maximum extract from the plant under investigation in comparison to other solvents like benzene, chloroform and acetone used for extraction. The fruits of B. serrata give maximum yield (13.9±0.02 g/100g) when it is extracted with methanol and the least amount is observed with benzene. The differences in the extractive value of the plant materials may be due to the varying nature of the chemical components present and the polarities of the solvent used for extraction.

Table 2. Antioxidant activities of B. serrata using different solvents

Parameter studied

Benzene

Chloroform

Acetone

Methanol

Extractive value (%)

1.0±0.0001

1.45±0.002

3.65±0.03

13.9±0.02

Total phenolic  content (Gallic acid equivalent, mg/100gm Plant material)

63.84±1.10

76.02±1.59

83.71±0.97

545.25±0.77

Total Flavonoid content (Rutin equivalent mg/100gm Plant material)

28.64±0.09

30.33±0.11

50.98±0.06

110.51±0.08

Total flavonol content (Quercetin equivalent mg/100gm Plant material)

69.37±1.11

71.75±0.71

99.81±0.86

223.11±0.55

Reducing power (Ascorbic acid equivalent mg/100gm Plant material)

24.6±0.95

17.71±1.14

29.09±0.53

131.48±0.27

DPPH radical scavenging activity (IC50 mg dry extract)

0.08±0.01

0.36±0.002

0.2±0.01

0.025±0.0003

ABTS radical scavenging activity (IC50 mg dry extract)

0.36±0.002

0.24±0.008

0.11±0.02

0.02±0.00001

Each value in the table was obtained by calculating the average of three experiments and data are presented as Mean ± SEM

Total phenol, flavonoid and flavonol content in the extract

The screening of the benzene, chloroform, acetone and methanol extracts of the plants revealed that highest amount of phenolic compounds, flavonoid and flavonol were detected in the methanol extract of the plant.  The results strongly suggest that phenolics are important components of these plants. The other phenolic compounds such as flavonoids, flavonols, which contain hydroxyls are responsible for the radical scavenging effect in the plants. According to our study, methanol was the most suitable solvent to isolate the phenolic compounds and benzene, chloroform and acetone are the best solvent to isolate the flavonoids and flavonols from the plant materials. The total phenolic component exhibited antioxidant activity through adsorption and neutralization of the free radicals, whereas flavonoid and flavonol showed antioxidant activity through scavenging or chelating process (Florence et al., 2011; Pourmorad et al., 2006). The high content of the phenolic compounds in B. serrata can explain their high radical scavenging activity. In this study the methanol extract of B. serrata showed potent antioxidant activities using DPPH and ABTS assay. The IC50 value of DPPH assay of B. serrata was found to be higher than that of ABTS assay which showed more antioxidant activities. The high radical scavenging property of this plant may be due to the presence of hydroxyl groups that can provide the necessary component as a radical scavenger.

The antioxidant activities of the extractive solution represent an important parameter to evaluate the biological property of the plant. Therefore, it is necessary to characterize and quantify the important compounds like phenolic acids and flavonoids present in the plant and also to validate the method of separation and identification of active constituents.

The HPLC analysis showed (figure1, table 3) the presence of good amount of p-coumaric acid (4.33±0.03 mg/100gm plant material) in the methanol extract of B. serrata.  Due to the presence of p-coumaric acid, the plant is believed to have antioxidant behavior thereby reducing the formation of carcinogenic nitrosamines in the stomach (Ramadoss et al., 2015).

Table 3. Phenolic acid and flavonoid content in B. serrata by HPLC

Phenolic acids/flavonoids

Amount (mg/gm dry plant material)

Phenolic acids/flavonoids

Amount (mg/gm dry plant material)

Phenolic acids/flavonoids

Amount (mg/gm dry plant material)

Gallic acid

0.48±0.008

Caffeic acid

2.51±0.06

Rutin

0.62±0.002

Protocatechuic acid

ND

Syringic acid

0.87±0.001

Ellagic acid

3.31±0.05

Gentisic acid

ND

p-Coumaric acid

4.33±0.03

Myricetin

3.89±0.003

p-Hydroxy benzoic acid

ND

Ferulic acid

7.74±0.0003

Quercetin

9.17±0.001

Catechin

ND

Sinapic acid

1.05±0.001

Naringenin

ND

Chlorogenic acid

1.83±0.003

Salicylic acid

ND

Apigenin

1.53±0.002

Vanillic acid

ND

Naringin

0.57±0.004

Kaempferol

ND

Each value in the table was obtained by calculating the average of three experiments and data are presented as Mean ± SEM

Figure 1. HPLC chromatogram for the quantification of phenolic acids in B seratta

 

 

The plant was found to contain a very good amount of chlorogenic acid (1.83±0.003 mg/100gm) which is responsible for   reducing hepatic triglycerides levels, thus resulting in weight loss. It also decreases proliferation of new fat cells through its antioxidant effects (Viviane and Adriana, 2009).

One of the important phenolics, ferulic acid which is detected in the methanol extract of the plant in our study and regular intake of the vegetable leads to lower cholesterol level in serum and increases sperm viability (Mussatto et al., 2007).

A very significant amount of sinapic acid was detected in the plant under investigation and consumption of this plant would be useful for health promotion because it showed antioxidant, anti-microbial, anti-inflammatory, anticancer, and anti- anxiety activity (Sepulveda et al., 2011).

A very good quantity of quercetin, detected in B. serrata was comparable to the same in apple (0.021 mg/gm), lettuce (0.011 mg/gm) and tomato (0.055 mg/gm) and this is reported to display anti-histamine, anti-cancer as also anti-inflammatory activities (Wach et al., 2007).

The HPLC analysis of the methanol extract of B. serrata showed the presence of good amount of myricetin (3.89±0.003mg/100gm DPM), which is widely available in fruits, vegetables, tea, berries and red wine and reported to be useful for the prevention of diabetes mellitus and diabetic complications (Chaudhury et al., 2018).

Biosynthesis of gold nanoparticles using methanol extract of B. serrata

The fruit extract of B. serrata in methanol is a rich source of different types of plant secondary metabolites such as polyphenolic compounds (545.25 ±0.92 mg GAE/100 g dry material), flavanoids (110.51±0.08 mg/100 g dry extract), flavonols (223.11±0.59 mg/100 g dry extract) etc. HPLC analysis of the fruit extract carried out in our laboratory also supported the presence most of the compounds. The fruit extract of the investigated plant, rich in polyphenolic compounds, can be utilized for the synthesis of AuNPs from HAuCl4.

Synthesis of BS-AuNPs and study of its Surface Plasmon Resonance spectroscopy

Antioxidants including polyphenols are well known for their use in the facile synthesis of metal nanoparticles under very mild condition.  As the fruit extract of B. serrata was rich in easily oxidizable plant secondary metabolites including polyphenols, we felt that fruit extract of B. serrata can be utilized for the green synthesis of BS-AuNPs at room temperature.  To test this, we treated the aqueous solutions of the fruit extract contained in vials with HAuCl4 solution (Figure 2). Violet to pinkish red coloration appeared after 5 minutes indicating the formation of BS-AuNPs.  The intensities of the colors increased on standing the solutions at room temperature for several hours and then remained constant and the BS-AuNPs once formed were stable for several months at room temperature (Majumdar et al., 2013; Barai et al., 2018).  The HAuCl4 showed a strong peak at 243 nm and a shoulder peak at 298 nm. This was due to the charge transfer interactions between the metal and the chloro ligands (figure 2a).  The intensities of these two peaks decreased with increasing concentration of the fruit extract of B. serrata and new peaks appeared around 532 nm.  This is due to surface Plasmon resonance (SPR) of the BS-AuNPs, a phenomenon arising due to collective oscillation of the conduction band electrons interacting with the electromagnetic component of the visible light. With increasing the concentration of the fruit extract, a blue shift of the SPR band was observed due to the formation of smaller sized BS-AuNPs.  The shoulder peaks observed in the 270-275 nm regions of BS-AuNPs colloids were due to the formation of quinone moiety formed by the oxidation of the phenolic compounds.

Figure 2. UV-visible spectra of (a) HAuCl4 (0.67 mM), (b-e) AuNPs at 100, 200, 300, and 400 mgL -1 concentrations of fruit extract respectively. Inset: Photograph of the vials containing (a) HAuCl4 (0.67 mM) solution, (b-e) colloidal BS-AuNPs at 200, 400, 600 and 800 mgL -1 of fruit extract of B. serrata respectively (after 24 h of mixing). 

 

 

Mechanism of the formation of Stabilized AuNPs

Fruit extract of B. serrata is rich source of different types of phytochemicals including polyphenols, flavanoids, flavonols, etc. The o-dihydroxy compounds present in the fruit extract can form a five membered chelate ring with the Au (III) ions.  Au (III) ions having a very high reduction potential can be reduced to Au (0) with concomitant oxidation of the polyphenols to corresponding quinones. The freshly generated Au (0) atoms in the reaction mixture can collide with each other forming AuNPs which are stabilized by the concomitantly formed quinones, polyphenols and other coordinating phytochemicals. The steric bulk of the backbone of the benzoquinones derivative and other phytochemicals wrapping around the nanoparticles provide robustness against further aggregation of the stabilized BS-AuNPs (Figure 3).

Figure 3. Mechanism of the formation and stabilization of BS-AuNPs by the phytochemicals present in the fruit extract of B. serrata taking myrecetin as a representative polyphenol.

 

HRTEM, SAED, EDX and XRD studies

High resolution transmission electron microscopy (HRTEM) was carried out to study the size distribution, shape and morphology of the BS-AuNPs formed at a particular concentration of the fruit

extract of B. serrata. BS-AuNPs of spherical, triangular, tetragonal, pentagonal and hexagonal shapes were observed (Figure 4a, b). The average size of the BS-AuNPs formed at 800 mgL-1 concentration of the fruit extract was 10.8 nm (figure 4c). The polyphenolic compounds, quinone and other chelating phytochemicals present in the fruit extract could effectively stabilize the smaller sized BS-AuNPs.  Formation of the BS-AuNPs was also confirmed from SAED and EDX analysis which showed the presence of Au along with C from the stabilizing organic ligands (Figure 4d, e).

A colloidal BS-AuNPs sample was coated over a glass plate, the volatiles were removed and X-ray diffraction analysis of the dried BS-AuNPs sample was carried out. The reflections of the planes (111), (200), (220) and (311) at 2q = 38.12°, 44.21°, 64.81°  and 77.75° respectively (Figure 5) resembled the characteristic reflections of crystalline metallic face centered cubic Au (JCPDS file no. 04-0784). The higher intensity of the (111) plane indicates predominant orientation of this plane compared to the other planes.

Figure 4. (a,b) HRTEM Images of BS-AuNPs obtained from the fruit extract of B. serrata at 800 mgL-1, (c) Histogram of (a), (d) SAED of stable gold nanoparticles obtained from the fruit extract of B. serrata at 800 mgL-1, (e) EDX stable gold nanoparticles obtained from the fruit extract of B. serrata at 800 mgL-1.

 

 

 

Figure 5:  XRD pattern of stable BS-AuNPs synthesized using the leaf extract of  B. serrata (800 mgL-1).

 

 

Conclusion

The present investigation revealed the presence of good amount of protein, fat, carbohydrate and fibre in this plant which could provide essential nutrients required for maintaining normal body function. The nutritional property of this plant was similar to and also sometimes better than the common vegetables. The fruits of the plant were also found to be a significantly useful source of various minerals like Na, K, Ca, Fe, Cu, Mg and Zn.  The fruits of this plant could be used for the nutritional purpose of human being due the presence of various phenolic acids and flavonoids  and adequate protection may be obtained against diseases arising from malnutrition. The presence of significant amount of respective bio-active components in this plant under study and variation of quantity determined ensures its usefulness for the synthesis of gold nanoparticles, without requirement of any reducing agent. The high amount of phenolic acids present in B. serrata acted as an electron donor system and ligating agents to form stabilized nanoparticles. Therefore, using this plant extract will be a new and favourable alternative to the current processes to produce metallic nanoparticles in large scale without generating any toxic by-products.  HRTEM studies revealed the mostly spherical shape of the AuNPs of average size of 10.8 nm. As, B. serrata is non-toxic and edible, the gold nanoparticles synthesized by the green synthetic method utilizing the active ingredients present in the plant extract will be useful for various biomedical as well as nano-scientific applications.

Acknowledgements

The Authors TS and KC owe deep gratitude to Dr. P. Singh, Director, Botanical Survey of India, Kolkata for extending necessary scientific facilities.  SNH, ACB and SD thank to UGC, New Delhi for a research fellowship. BGB thanks Science and Engineering Research Board (SERB), India (ref. EMR/2016/001123), India Srilanka project (DST/INT/SL/P25/2016), UGC-MRPMAJOR-CHEM-2013-35629, UGC-SAP DRS II and DST-FIST New Delhi and Vidyasagar University for financial support and infrastructural facilities.

Conflicts of interest: We have no conflict of interest.

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