INTRODUCTION:

The Sapindus mukorossi, often known as soapnut, is a member of the Sapindaceae family. It is medicinally use as an expectorant, a contraceptive, and to treat migraines, chlorosis, excessive salivation, and epilepsy¹. It is a well-known component of ayurvedic shampoo, cleansers, and medicines for treating eczema, psoriasis, and eliminating freckles.

It also has a mild insecticidal effect and is traditionally used to get rid of lice from the scalp. Triterpenoid saponins, primarily three types—oleanane, dammarane, and tirucullane—are the principal substances identified from Sapindus Mukorossi. Numerous pharmacological effects of this plant, such as its antibacterial, cytotoxic, molluscicidal, insecticidal, pesticidal, and fungicidal activities, have recently been studied².

Nanotechnology is the creation, manufacturing, characterization, and manipulation of materials, devices, or structures with at least one dimension of between 1 and 100nm in length. The physical and chemical properties of materials with particle sizes below this cutoff are very different from those of macroscale materials made of the same constituent.

Due to the growing need for ecologically acceptable material synthesis techniques, synthesizing nanoparticles has drawn more interest in the last ten years. recently has the biosynthetic method using plant extracts drawn interest as a quick and practical substitute for chemical processes and physical methods manufacturing metal nanoparticles³. The food and health sectors are growing more and more dependent on nanotechnology. Through bioactive nano-encapsulation, promising outcomes and applications are already produced in the fields of nutrient and medication delivery systems, as well as the creation of better, safer, and more effective therapeutic formulations.

The unique optical, thermal and electrical properties of silver nanoparticles (AgNPs), which improve electrical conductivity, near-infrared absorption, and efficient charge separation, make them of contemporary interest. Due to their abundant availability and diverse range of reducing metabolites, plant extract is favored over other biological sources. The biosynthesis of AgNPs from plant sources has various benefits over conventional synthesis methods, including cost effectiveness, environmental friendliness, and the absence of harmful chemicals and high pressure, energy, and temperature^4,5. Using phytochemicals, the process of plant-assisted reduction is the fundamental mechanism behind the process. The primary phytochemicals at play are flavones, terpenoids, ketones, amides, aldehydes and carboxylic acids. Quinones, organic acids, and flavones are polar phytochemicals which cause the ions to be reduced right away. Research showed that xerophytes contain emodin, an anthraquinone which undergoes tautomerization to form silver nanoparticles. It was found that the three types of benzoquinones found in mesophytes are cyperoquinone, dietchequinone, and remirin. According to one theory, phytochemicals are responsible for ion reduction resulting formulations of silver nanoparticles⁶. The main mechanism is the reduction of the ions, even if each plant's extract mechanism differs depending on the phytochemicals involved.

MATERIAL AND METHODS:

Collection of crude Drug:

Sapindus mukorossi fruits were picked, cleaned in tap water to get rid of the dust, then immersed insaline water (1%) for 5 minutes to get rid of the germs. Finally, the fruits were dried in the shade to preserve their rich vitamin content includinglight sensitive components. The plant material was allowed to dryover sterile and ventilated room. The fruits and seeds were separated, and the nice fruits were visually distinguished. The dried and separated seeds were manually crushed into small, coarse pieces and kept in an airtight container that was shielded against light and humidity⁷.

Physiochemical Parameters:

Moisture Content:

The loss on drying method can be used to determine the moisture content. The parameter establishes how much moisture and volatile substances are contain in a specific sample. A Petri dish containing 2gm of powdered bark and leaves has been used, and it was then heated to 105°C in a hot air oven. LOD was then determined⁸.

Extractive values:

The amount of a medicine that can be extracted by a solvent is measured by its extractive value. The extractive value depend on solubility in water, alcohol, ethyl acetate, or chloroform⁹.

Successive extraction:

In a series of extraction methods, chloroform, methanol, ethyl acetateand water are used to extract compounds from powdered plant material. The type of solvent used depends about how polar the compounds in the plant material seem to be.

For example, chloroform and hexane are non-polar, while ethyl acetate is intermediately polar and methanol and water are highly polar. For consecutive extraction, the Soxhlet apparatus for the hot percolation process was utilized. Here, a medicine or other item that is recommended in a monograph is continuously extracted¹⁰.

Preparation of extracts:

Four times, for an hour each at 70°C, methanol, ethyl acetate, chloroform, and water were used to extract the powdered fruits of Sapindus mukorossi. In a distillation assembly, all of the extractive was filtered by muslin fabric, collected, and allow to dry via vacuum. During the water extraction process, foam was created, hence 2 to 3ml of paraffin liquid was utilized. It passed by the muslin fabric to be filtered. For the purpose of separating the liquid paraffin, the water extract was collected, agitated with hexane, and then let to remain for some time. After that, it is vacuum-dried, weighed, and the percentage was estimated¹¹.

Phytochemical Screening:

These screening involves the analysis of plant extract. Phytochemical screening shows the presence of various phyto-constituents in the plant extract such as Chloroform, Hexane, Methanol, Ethyl acetate, and water, using the Hot percolation (Soxhlet apparatus). The active constituents are then subjected to qualitative screening for the characterization of Alkaloid, Saponins, Steroids, Flavonoids, Phenolics, Glycosides and proteins. The table 1. Shows the procedures for determining the Phyto-chemical constitutes of plant extract¹².

Synthesis of AgNPs:

AgNPs were synthesized using water and methanol fruit extract of Reetha were carried out by addition of 25ml and 75ml broth of fruit extract to a reaction mixture containing 1M AgNO₃ and 0.625% ammonia solution and left for 48hrs in dark place with regular interval exposing to light. The prepared silver nanoparticles were separated by using 0.22µm membrane filter paper¹³.

Characterization of Sapindus mukorossi Extract AgNPs:

Fourier Transform Infrared spectroscopy:

FTIR studies within a wavelength range of 4000–500 cm-¹was carried out in order to identify the functional moiety of bioactive agent of Sapindus mukorossi extract responsible for the reduction of Ag+ ions into Ag and AgNPs stabilization¹⁴.

Surface Morphological Studies:

Scanning Electron Microscopy:

SEM was performed for analyzing surface morphology and shape of the synthesized AgNPs. To convert the AgNPs sample electrically conductive, a thin film of gold (3-5nm) was applied for 75 seconds at 40 W in a vacuum, after which the sample was positioned on the stubs by double-sided adhesive tapes. The images were taken with a 20 kv excitation voltage and magnifications of 4.79 and 6.32 KX¹⁵.

X-Ray Diffractometry:

XRD pattern of AgNPs using an X-ray diffractometer (EXPERT-PRO) was performed. Samples were analyzed in the 5–30° range on the 2θ scale under Cu Kα radiation at a voltage of 40kv and a current of 40mA¹⁶.

Antibacterial Activity:

Preparation of Sabourand Dextrose Agar (SDA) media:

Weighed amounts of dextrose (40mg), peptone (10mg), and agar (15mg) were dissolved into distilled water. Agar was dissolved after the mixture was warmed and allowed to sterilized in autoclave for 15mins at 121°C and 15lbs. Under aseptic conditions, the sterilized media (20ml) was put onto sterilized Petri plates, allows them to settle on flat surface¹⁷.

Preparation of Antibacterial Solution:

Dimethyl formamide was used to dissolve each component. Drug controls, accurately depict the inhibition zone for the control and the extract were employed. To assess the antifungal activity of the extract, 0.1mg/ml of the substance was ingested. After the incubation, inhibition zone was measured in mm and the concentration gradient produced by the extract diffused into medium¹⁸.

Test Cultures:

E. coli and Streptococcus aureus strains were used for In-vitro antibacterial activity.

Inoculum Preparation:

The pre-cultured fungi were housed in a BOD incubator at 25°C for the duration of an overnight pre-culture in nutrient-filled bottles. One loopful of a culture broth of the test organism that had been cultivated overnight was used as the inoculum for determining the sensitivity pattern. The inoculum was typically approximately the size of a 2mm diameter standard loop.

Experimental procedure:

A standard inoculum of the test microorganism is used to inoculate agar plate. The test substance is then added to filter paper discs (approximately 5mm in diameter) at the specified concentration and spread on the agar surface. At 25°C, the plates were put in an incubator. The zone diameter was measured in mm a few days following fungal development¹⁸.

Screening of antifungal activity against bacteria:

In order to do screening, various solvent extract concentrations are used. Drug plates and varied sterile Sabourand dextrose agar media were prepared, along with various compounds of varying concentrations. One gram-negative bacteria (E. coli) and one gram-positive microbe (Streptococcus aureus) were used for the initial screening. All of the microbes were spot-inoculated into drug plates that contained various chemical concentrations. The drug plates were incubated at temperature 22°C for 24-hours and again for three more days to detect microorganisms. Different plates were examined to determine whether bacterial growth had occurred after 24hours of incubation. According to NCCLS (National Committee for Laboratory Standards) criteria, the extract's minimum inhibitory concentration (MIC) was assessed against various fungus strains^19,20.

RESULTS:

Physiochemical Parameter:

In Table no. 1 shows the results of all physiochemical tests parameters of water-soluble extracts and ethanol soluble extracts.

Table 1: Physiochemical parameters tests results

Physiochemical Tests	Value (%w/w)	Water-soluble extractive value	Value (%w/w)	Ethanol-soluble extractive	Value (%w/w)
Ash	2.2	Cold maceration	66.5	Cold maceration	48.9
Acidic-insoluble ash	0.012	Hot percolation	76.0	Hot percolation	63.14
Loss on drying	7.1	-	-	-	-

Table 2: Percentage extractive value results

S. No	Solvents	Extract Colour	Value (%w/w)
1.	Chloroform	Greenish yellow	22.43
2.	Ethyl acetate	Pale yellow	13.22
3.	Methanol	Light brown	46.4
4.	Water	Bark brown	70.11

Table 3: Phytochemical screening results

S. No	Tests	Extract of Chloroform	Extract of Ethyl acetate	Extract of Methanol	Extract of Water
1.	For carbohydrate
	Molisch test	+	+	+	+
	Fehling Test	+	+	+	+
2.	Proteins
	Biuret Test	-	-	-	-
	Millon’s Test	-	-	-	-
3.	For Phenolic compounds/ Tannins
	Ferric chloride solution Test	-	-	-	-
	Lead acetate Test	-	-	-	-
4.	For Steroids/ Triterpenoids
	Salkowski reactions	-	-	-	-
	Liebermann – Burchard Reactions	+	+	+	+
5.	For Flavonoids
	Shinoda test	-	+	+	-
	Lead acetate solutions	-	+	+	-
6.	For Alkaloids
	Mayer’s test	-	-	-	-
	Hager’s test	-	-	-	-
	Dragendorff’s test	-	-	-	-
7.	For Saponins
7.	Foam test	+	+	+	+
8.	For Glycosides
	Bontrager’s test	-	-	-	-
	Modified Bontrager’s test	-	-	-	-

Table 4: Results of total saponin results and percentage yield of silver nanoparticles

Sample	Total Saponins percentage results			Percentage Yield of Silver Nanoparticles
Sample	1 Method	2 Method	3 Method	Formulation	% Yield
Aqueous	60	-	59	Water Extract 25%	58.34%
Alcoholic	34.11	15.12	32.51	Water Extract 75%	77.46%
Ethyl acetate	65.45	0	63.11	Methanol Extract 25%	49.67%
Chloroform	75.89	0	72.23	Methanol Extract 75%	69.32%

Percentage Extractive Value:

Table no. 2 shows the percentage extractive values by successive solvent extraction method.

Phyto-Chemical Screening Test Results:

Table no. 3 shows the result of phytochemical screening, which indicates the Phyto-chemical contents of plant extract, (+) indicates presence, (-) and indicates absence of content of extract.

Total Saponins by Gravimetric Method: Total saponins three methods were used and percentage concentration of total saponin in different extract sample are mentioned in table 4.

Percentage Yield of Silver Nanoparticles: Silver Nanoparticles are prepared using water and methanol extract at different concentration i.e., 25% and 75%, the results are mentioned in Table 4.

Fourier Transform Infrared Spectroscopy:

As observed in Figure. 1 (A), The extract's FTIR spectrum showed a significant broad spectrum at 3417.12 cm^-1 shows to the O-H stretching vibration. The C–H stretching along with wagging vibrations belong to the maxima at 2854.98 and 1384.28 cm^-1, respectively. The vibrations attributed to C = O stretching and C-O bending are detected at 1725.79 and 1249.74 cm^-1, respectively. The C = C alkenyl of aromatic ring and the amide ring C = Ostretching is responsible for the peak at 1632.27 cm^-1, whereas the C-O-C bending is responsible for the peak at 1055.71 cm^-1. The existence of residual organic molecules on the AgNPs' surface during the reduction reaction and purification is indicated by equivalent absorption peaks at around 3445.12, 2917.23, 1715.71, 1642.23, 1382.23, and 1065.34 cm^-1. Nonetheless, the development of silver-carboxylate bonds with hydrocarbon chains via hydrophobic forces could be responsible for a significant reduction in the peak intensity and position for AgNPs samples at 2917.23, 1715.71, and 1243 cm^-1.Furthermore, a strong peak at around 1642.23 cm^-1due to C = O of the amide group or the C = C of the aromatic ring may indicate that saponin as well as flavonoidsare involved in the reduction process.

X-Ray Diffractometry:

As shown in Figure.1 (B), the AgNPs diffraction pattern showed sharp peaks at 2h = 39.12 (111), 43.12 (200), 65.02 (220), and 78.67 (311) indicating highly crystallized AgNPs. Results shows no impurities peak, confirming that the synthesized AgNPs were purely of silver metal.

Figure. 1 (A) FTIR Images of (a) Synthesized Sapindus mukorossi extract AgNPs. (b) Sapindus mukorossi extract. Figure (B) XRD of Synthesized Sapindus mukorossi extract AgNPs.

Surface Morphological and structural studies:

Scanning Electron Microscopy:

Figures 2(A) and (B) demonstrate the shape and size of the synthesized AgNPs, which were found to be relatively spherical-shaped nanoparticles with a comparatively uniform diameter. AgNPs have been reported in a variety of shapes by various studies, such as spherical,pentagonal, triangular, hexagonal and cuboidal geometries. The spherical AgNPs that were produced in this investigation therefore match the forms that are anticipated for AgNPs. Through the exchange of electron from functional groups, the phytochemicals included in plant extract, such as saponins, are in charge of reducing Ag+ ions to Ag. The naturally occurring surfactant saponin molecules were then encapsulated in the as-reduced AgNPs. The steric hindrance mechanism may influence the kinetics of reaction and also inhibit AgNPs aggregation by collision.

Figure 2: SEM Images of Synthesized Sapindus mukorossi extract AgNPs. (A) Surface view at 4.79 KX (B) Surface view at 6.23 KX

Antibacterial Activity:

Various solvent extract concentrations are used to determine the antibacterial activity. Gram-negative bacteria (E. coli) and Gram-positive microbe (Streptococcus aureus) were used for the initial screening. All of the microbes were spot-inoculated into drug plates that contained various chemical concentrations. Different plates were examined to determine whether bacterial growth had occurred afterincubation (24hr). In Figure 3 (A) and (B), shows the antibacterial activity of E. coli and Streptococcus aureus respectively. Table 5 shows the observation of zone of inhibitions of different plant extract concentrations.

DISCUSSION:

The physical appearance of the nanoparticles found as the brownish-black with sweet smell. The total ash was found to 2.2% w/w, acidic -insoluble ash was fund to be 0.012% w/w and loss on drying was obtained 7.1% w/w. The percentage extractive value was found to be for chloroform, ethyl acetate, methanol and water was 22.43, 13.22, 46.4 and 70.11% w/w/ respectively. The total saponin value was determined by gravimetric methods, in which chloroform extract was maximum saponin content that is 75.89% w/w. The phytochemical screening confirms the presence of carbohydrates, steroids, flavonoids and saponins in plant fruit extract. The solubility of the drug was found to be practically soluble in water and methanol. The yield of the nanoparticles is more in water than methanol, the methanol-based nanoparticles are more stable than the water-based nanoparticles. The extract contained bioactive agents that acted as reducing and capping agents for the synthesis of AgNPs. The synthesized AgNPs were characterized via SEM, XRD and FTIR studies. The results indicated that spherical AgNPs, spherical and capped with saponins and other organic compounds from the Sapindus mukorossi fruit extract. The methanol-based nanoparticles showed greater shelf life than water-based nanoparticles. The methanol-based nanoparticles show moreantibacterial response against Streptococcus aureus media and water-based nanoparticles shows more antibacterial response against E. coli bacteria media.

CONCLUSION:

The Phyto-based drugs exhibit good medicinal properties with less side-effects as compare to synthetic based drugs. The reetha is known for its many properties such as foaming agent, antibacterial activity etc. In this work, we have performed a green chemistry approach for the synthesis of silver nanoparticles using Sapindus mukorossi fruit pericarp extract for development the new antibacterial drugs as the humans getting resistant to the antibacterial drugs. So, with the known potent antibacterial silver nanoparticles which enhance the antibacterial properties of the reetha extract.The antibacterial results show that the prepared AgNPs were effective in inhibiting growth of both S. aureus and E. coli bacteria, and hence they could be potential applications in bio-related field.

ACKNOWLEDGEMENT:

I would like to acknowledge all the co-authors who have rendered their invaluable contribution in successful completion of my research work. The authors would especially like to thank Prof. (Dr.) Dharam Buddhi, Vice Chancellor of Uttaranchal University, and Mr. Jitender Joshi, Chancellor, for their valuable support. The authors would also like to thank Prof. (Dr.) Vikash Jakhmola, Dean of UIPS, for his inspiration and motivation. We are grateful to Uttaranchal University-Dehradun, India's Division of Research and Innovation (DRI) along with Central Instrumentation Facility (CIF) for providing the facilities needed for the research experiment. We are also thankful to Uttarakhand State Council for Science and Technology UCOST for constant support during the research work.

CONFLICT OF INTEREST:

None.

FUNDING SOURCE:

This work is done under the seed money project, funded by the Division of Research and Innovation (DRI), Uttaranchal University Dehradun (India), the grant number of funding sources is UU/DRI/SM/2022-23/005.

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Received on 28.12.2022 Modified on 10.06.2023

Research J. Pharm. and Tech 2023; 16(12):5643-5649.

DOI: 10.52711/0974-360X.2023.00912

S. No.	Antibacterial activity against E. coli		Antibacterial activity against Streptococcus aureus
S. No.	Sample taken	Observation (mm)	Sample taken	Observation (mm)
1.	Methanol (25% extract)	18	Methanol (25% extract)	19
2.	Methanol (75% extract)	25	Methanol (75% extract)	26
3.	Water (25% extract)	26	Water (25% extract)	22
4.	Water (75% extract)	15	Water (75% extract)	17