Green Synthesis of Silver Nano Particles from Cheilocostus Speciosus (J. Konig) C. Specht and Enriching Yogurt with Alpha Amylase Activity

Diabetes mellitus is known as a serious, complex condition, a group of disorders that have hyperglycemia and glucose intolerance as their hallmark, due to insulin deficiency or to the impaired effectiveness of insulin’s action, or both [1]. It is also described as a chronic disease associated with elevated levels of glucose in the bloodstream which causes glycation of the body protein leading to severe complications [2]. Complications include neuropathy (gradual damaging of the nerves), retinopathy (gradual damaging of the eyes), nephropathy (gradual damaging of the kidneys), polyuria, visual blurriness, disproportionate thirst, fatigue, irritability, weight loss and hypertension [3]. 

Yogurt is one of the oldest and most important fermented dairy products because of its high nutritional value. It is associated with many health benefits, from relieving diarrhea to reducing the risk of colorectal cancer [4,5]. These health benefits are thought to be mediated through maintaining the appropriate balance of the gut microbiota and regulating the microenvironment of the intestinal immune system [6,7]. 

Working with these extremely small structures is very much interesting due to its unique properties. To minimize drug degradation and loss, to prevent harmful side-effects and to increase drug bioavailability and the fraction of the drug accumulated in the required zone [8,9]. The use of plant products for the synthesis of nanoparticles adds a new dimension for modern nano biotechnology. 

Costus speciosus is distributed in the tropical and subtropical regions of Asia, Africa, and Americas. The underlying rhizome is the perennating organ of C. speciosus which is differentiated into nodes and internodes and aboveground vegetative stage lasts for about 7-8 months. The leaves are spirally arranged on the erect or ascending stem, manifested by broad, lance shape and silky instances on dorsal site. The flowers are white in color, clustered in terminal globose heads, with large and shiny brown or red bracts. Plant contains several bioactive compounds, such as polyphenols, flavonoids, and alkaloids. That provide major source of molecular properties due to presence of natural compounds. This plant is reducing blood sugar level to a significant amount, more so when consumed on empty stomach. Adding to the diet increases the secretion of insulin and help manage diabetes better. This can be helpful for people with diabetes to assist in reversing the oxidative stress of the liver, of the liver, pancreas, and kidneys. The plant also has diuretic, antimicrobial, and cancer preventing properties. The present study green synthesis of silver nanoparticles from the methanolic extract of Costus speciosus rhizome. Preparation of yogurt with Costus speciosus rhizome powder and silver nanoparticles and evaluation the alpha amylase activity.

Collection of Plant Material 

C. speciosus rhizome collected from Sardar Patel University Botanical Garden. Plant material was collected between November and December 2023. Plant material of healthy and disease-free plants was used to synthesis of silver nanoparticles and yogurt.

Drying and Grinding the Plant Material 

The plant material was collected and washed under running tap water to remove dust particles. Then the plant material was kept to dry room temperature with adequate ventilation. Dry conditions are essential to prevent subsequent degradation of metabolites. Protection from direct sunlight is advised to minimize chemical reactions induced by ultraviolet rays [10]. After drying, plant materials were grounded into powder form. Grinding of plant materials into smaller particles facilitates subsequent extraction processes, increasing the surface area. The powder was passed through a 60-mesh sieve to get a fine powder.

Preparation of Plant Extract by Soxhlet Method

C. speciosus rhizome 5gm powder was extracted with 250ml methanol for four hours. After the extraction procedure the solvent were evaporated under room temperature [11]. 

Green Synthesis of Silver Nanoparticles from C. Speciosus Rhizome 

AgNo3 nanoparticles were successfully synthesized using plant rhizome extracts by adopting the above procedure with slight modification [12]. 

Separation of Silver Nanoparticles 

The synthesized silver nanoparticles were separated by centrifugation using a REMI centrifuge at 10,000rpm for 15min. The supernatant liquid was re-suspended in the sterile double distilled water. The process was carried out thrice to get rid of any on co-ordinated bio molecules. After, the desired reaction period, the supernatant liquid was discarded, and the pellets were collected and stored at 4°C for further use [12]. 

Characterization of Silver Nanoparticles 

• U-V Visible Spectrophotometer 

U-V visible spectrophotometer was performed for the detection of AgNPs. Synthesized by plant rhizome material λ max around 420nm indicates the formation of AgNPs [13].

• Fourier Transform Intra Red Spectroscopy (FT-IR) 

To identify the biomolecule associated with the synthesis of nanoparticles by plant rhizome mediated was performed by using FT-IR [14].

• XRD Analysis 

Further characterization of AgNPs was carried out through XRD (D8 Advance, Bruker) equipped with Cu-Kα radiation; a crystal monochromator employing wavelengths of 0.1541nm in a 2θ range from 20° to 80° [15]. 

• Preparation of Yogurt 

Pour milk of choice into a double boiler and heat to 180°C after follow the method reported. Refrigerate yogurt immediately once the yogurt has congealed to a jell-like consistency. Rapid chilling stops the development of acid [16].

In -Vitro Alpha Amylase Inhibitory Activity

• Assay of Alpha Amylase Inhibitory Activity 

When amylase acts on starch its converter into glucose units. The resultant glucose unit react with 3, 5-Dinitrosalicylic Acid (DNS) in alkaline solution to give rise to orange coloured complex, which can be measured at 540nm. Control was prepared using the same procedure by replacing the inhibition percent of alpha- amylase enzyme was calculated using the following equation.

• α-Amylase Inhibition Screening Assay 

In this assay, 200μl of α - amylase enzyme and 0.1 to 1ml of plant extract, nano particles, and yogurt (one by one) mixture were mixed and incubated at 37°C for 45min. After, incubation the mixture was poured into the well made in the petri plate containing 3% agar (w/v) and 1.2% starch (w/v). Plates could stand for 3 days at 25°C and then flooded with iodine solution and allowed to stand for 15min. The diameter of zone of starch hydrolysis was measured. As a control, the enzyme was added into the well of the plate without plant extract. The % inhibition was calculated by following equation.

Nanotechnology is growing rapidly and has potential in range of commercial products. Any particle whose size ranges between 1nm to 100nm is called a nanomaterial. Synthesis of nanoparticle from plant is known as green synthesis of nanoparticles. It offers advantages of being cost effective, safe, and free of harmful chemicals. Synthesis of AgNo3 through this process was economical and rapid production. The AgNo3 exhibited substantial alpha amylase inhibition activity. 

Many plant extracts have been reported to have anti-hyperglycemic activity and are used in ayurveda for the treatment of diabetes. The presence study, an in vivo in inhibitory effect of plant extract, nanoparticles of plant extracts and its combination with yogurt on alpha amylase activity was evaluated. 

• Extractive Yield (%) of C. Speciosus 

Before evaluating the alpha amylase inhibition activity of C. speciosus plant knowledge about the yield of extract from each plant is important lower extract yielding plants are not commonly preferred by the pharmaceutical industry though they are rich in their potency [17]. So, the work was carried out with yield calculation. I have selected methanol for extracting the plant constituents. The yield of the methanol solvent is 0.0353%.

• Nanoparticles Formation 

There was a visible color change after the substrate was added to the plant extract. Initially the plant extract was colourless. Upon adding the silver salt, it turned brown. After 5hr, no significant colour change was observed. Increased concentrations of silver nitrate resulted in a brown solution of nano silver indicating the completion of reaction. Reduction of silver ions into silver nanoparticles using methanolic leaf extract of C. Speciosus on was evidenced by visual change of colour from colourless to brown colour which indicated the formation of silver nanoparticles due to the excitation of surface plasmon vibration in silver nanoparticles (Figure 1).

Figure 1: Nanoparticle formation of methanolic leaf extract of C. speciosus. 

Note: A: 0.01M AgNo3 and plant extraction at Zero minutes. B: 0.01M AgNo3 and plant extraction after two hours. C: 0.01M AgNo3 and plant extraction after four hours.

• UV-Visible Spectroscopy 

The UV-Visible spectroscopy was the preliminary technique for the characterization of the silver nanoparticles. The reduction of the pure Ag+ ions was monitored by measuring the UV-V is spectrum of the reaction medium at 5 hours (complete colour change) following the dilution of a small aliquot of the sample in distilled water. The UV-Vis spectral analysis was conducted using Shimadzu UV- Visible spectrophotometer, Model 1800 range between 200 and 600nm. The reduction of silver ions in the aqueous solution of nanoparticles in the solution could be correlated with the respective UV-Vis spectra of the colloidal solution which exhibited a strong absorption at 420nm as shown in figure 2. A typical peak was obtained due to the presence of surface plasmon resonance silver nanoparticles.

 Figure 2: UV-VIS spectrum of the silver nanoparticle.

• FT-IR Spectroscopy 

In FTIR analyses, Infrared light from the light source passes through a Michelson interferometer along the optical path. The Michelson interferometer comprises a beam splitter, moving mirror, and fixed mirror. The light beam split into two by the beam splitter is reflected from the moving mirror and fixed mirror, before being recombined by the beam splitter. As the moving mirror makes reciprocating movements, the optical path difference to the fixed mirror changes, such that the phase difference changes with time. The light beams are recombined in the Michelson interferometer to produce interference light. The intensity of the interference light is recorded in an interferogram, with the optical path difference recorded along the horizontal axis [18]. Figure 3 represent the FT-IR spectrum of rhizome extract of C. speciosus before adding into silver nitrate and show peaks situated at about 3410.97cm^–1, 2911.25cm^–1, 1671.19cm^–1, 1276.26cm^–1, 1051.48cm^–1,677.95cm^–1. Figure 4 represents of silver nanoparticles shows peaks situated at about 3445.76cm^–1, 2919.88cm^–1, 1660.37cm^–1, 1208.10cm^–1, 1075.47cm^–1.

 Figure 3: FT-IR spectrums of the plant rhizome extract of C. speciosus. 

 Figure 4: FT-IR spectrums of the plant rhizome nanoparticle of C. speciosus.

These peaks are known to associate with the figures 3 & 4, spectrum of the rhizome extract after adding into silver nitrate solution. It is showing functional group which involve in the biosynthesis of AgNPs from silver nitrate solution simple alkane, alcohol ether, carboxylic acid, and ester present (Tables 1 & 2). The alcohol ether, carboxylic acid, ester of these compound has a stronger ability to bind silver ions and may biosynthesis of AgNPs and act as reducing agent for the reduction of silver ions (Ag+) to silver nanoparticles (Ag°) the biological molecules such as secondary metabolites could possibly play major role in the synthesis and stabilization of the metal nanoparticles was proved [19].

Table 1: FT-IR Spectrum of the rhizome extraction of C. speciosus.

Table 2: FT-IR Spectrum of the rhizome nanoparticles of C. speciosus.

• Characterization by XRD 

The crystallinity of the synthesized silver nanoparticle using C. speciosus rhizome extract was examined through X-Ray Diffraction (XRD) (Figure 5). The size of the nanoparticles was calculated based on the Debye–Scherrer equation: (D = kλ/βcosθ). In the above equation: D represents particle diameter size, K: a constant with a value of 0.9, λ: X-ray source wavelength (1.5406nm), β and θ represent the FWHM (full width at half maximum), and diffraction angle concerning the lattice planes respectively. The average crystalline size was found to be approximately 58nm.

 Figure 5: XRD pattern of AgNPs synthesized from leaf extract of C. speciosus.

• Standard Alpha Amylase Inhibiter 

The inhibiters of alpha amylase bind to alpha bond of polysaccharide and stop the breakdown of polysaccharide in mono and disaccharide. For comparison with synthesis drug acarbose (standard drug for diabetes) was taken, it showed 58.45% inhibition effect on the alpha amylase activity at a concentration of 100µg/ml (Table 3). The solvent extracts of selected Costus speciosus rhizome showed higher alpha amylase inhibitory activity compared to acarbose.

Table 3: Alpha-amylase inhibitory effect of plant nanoparticles and plant nanoparticles with yogurt.

• Assay of Alpha Amylase Inhibitory Activity 

Alpha-amylase is an enzyme that hydrolyses to large alpha linked polysaccharide like starch and glycogen to yield disaccharides like maltose which will further hydrolyze by alpha- glycosidase to yield monosaccharides like glucose [20]. The inhibitors of alpha- amylase bind to the alpha- amylase bind to the alpha bond of polysaccharides and stop the breakdown of polysaccharide. 

Pancreatic α-amylase (E.C. 3.2.1.1), is a key enzyme in the digestive system and catalysis the initial step in hydrolysis of starch to maltose and finally to glucose. Degradation of this dietary starch proceeds rapidly and leads to elevated post prandial hyperglycemia. It has been shown that activity of Human Pancreatic α-amylase in the small intestine correlates to an increase in post-prandial glucose levels, the control of which is therefore an important aspect in treatment of diabetes. Hence, retardation of starch digestion by inhibition of enzymes such as α-amylase would play a key role in the control of diabetes. However, the discovery of specific high-affinity inhibitors of pancreatic α-amylase for the development of therapeutics has remained elusive. Inhibitors currently in clinical, use for example, acarbose, miglitol, and voglibose, are known to inhibit a wide range of glycosidases such as α- glucosidase and α-amylase. Because of their no specificity in targeting different glycosidases, these hypoglycemic agents have their limitations and are known to produce serious side effects. Therefore, the search for more safer, specific, and effective hypoglycemic agents has continued to be an important area of investigation with natural extracts from readily available traditional medicinal plants offering great potential for discovery of new antidiabetic drugs [21,22]. 

Hyperglycemia is the risk factor for the development of diabetes and its complications. Therefore, control of glucose levels in blood is a vital treatment for diabetes and the lessening of macrovascular and microvascular complications. One therapeutic approach is the reduction of postprandial hyperglycemic by suppressing hydrolysis of starch as PPA inhibition have been found effective in the control of diabetes mellitus. 

In the present study, plant rhizome extract of C. Speciosus and nanoparticles that show alpha-amylase inhibition activity. The inhibition effect of alpha-amylase of the methanol extract and their nanoparticle was studied. Plant extraction and plant extraction with yogurt showed maximum alpha-amylase inhibition activity at concentrations of 100µg/ml and 90µg/ml respectively (Figure 6). Percentage of inhibitory activity of alpha amylase by acarbose standard drug use for comparation (Figure 7). A strong absorption at 420nm as shown in figure 2. A typical peak was obtained due to presence of surface plasmon resonance silver nanoparticles [23]. 

Figure 6: % inhibitory activity of alpha amylase by Acarbose.

 Figure 7: % of inhibition of alpha-amylase by plant extraction, plant nanoparticles and yogurt combination

Plant extraction compared to plant extraction with yogurt showed better inhibition activity of the amylase. Plant extraction with yogurt more suitable for using for the controlling of the amylase inhibition and helpful in the control of the sugar level. But more focused requirement of the lower concentration also further check for inhibition of amylase. Ethanol extraction from rhizome reported by [24], Costus igneus, rhizome was used for the extraction process by ethanol. Dosage of different ranges were given such as (100mg, and 200mg) the analysis was based on observing the percentage reduction in glucose i.e. (63.30% and 68.26%) respectively. Our result show methanolic extract better than ethanolic extracts (Figure 7). 

Plant nanoparticle and plant nanoparticle with yogurt showed maximum alpha-amylase inhibition activity at concentrations of 100µg/ml and 10µg/ml respectively (Figure 7). Similar plant species reported by [25]. Costus igneus, leaves were used for the extraction process by methanol. Male wistar albino rats (wt. 150-200gm) were used to which dosage of different ranges were given such as (50mg/kg, 100mg/kg, and 200mg/kg). The analysis was based on observing the percentage reduction in glucose i.e. (52.44%, 64.81% and 69.08%) respectively (Figure 7). Our result show in the rhizome compared to the leaves are reduction of the glucose is very less. As per our result show the rhizome part are more beneficial compared to the leaves. It is more focus required for the animal experiment required for the comparison.

• Starch Iodine Assay

This study is based on the hydrolysis of starch by the alpha-amylase enzyme. The plant extraction and plant extraction with yogurt inhibit the alpha-amylase activity of starch hydrolysis. The enzyme which hydrolysed starch molecules gives monosaccharides, which give blue color when plates were flooded with iodine solution. The present study was therefore designed to investigate the plant extraction and plant extraction with yogurt relevant to the management of hyperglycemia by inhibition the alpha-amylase activity. In this study, plant extraction and plant extraction with yogurt were carried out by zone of clearance which was produced by starch hydrolysis. The selection of good plant extraction and plant extraction with yogurt for further analysis is depended upon the zone of clearance. 

Plant extraction and plant extraction with yogurt showed maximum alpha-amylase inhibition activity at concentrations of 30µg/ml and 30µg/ml respectively (Figure 8).

Figure 8: % of inhibition of alpha-amylase by starch assay method by plant extraction, plant nanoparticles and yogurt combination. 

The present study was investigating the plant nanoparticle and plant nanoparticle with yogurt relevant to the management of hyperglycemia by inhibition the alpha-amylase activity. In this study, plant nanoparticle and plant nanoparticle with yogurt were carried out by zone of clearance which was produced by starch hydrolysis. The selection of plant nanoparticle and plant nanoparticle with yogurt for further analysis is depended upon the zone of clearance. Plant nanoparticle and plant nanoparticle with yogurt showed maximum alpha-amylase inhibition activity at concentrations of 15µg/ml (Figure 8).

The result of the present study concluded C. speciosus rhizome was prepared using methanol solvent. Silver nanoparticles were successfully synthesized using plant extraction. The method described was highly efficient and cost effective to produce stable nanoparticles. Synthesis of AgNPs through this process was economical and rapid. The formation of silver nanoparticles was confirmed by UV-visible spectroscopy by peak formation at 420nm and XRD analysis show size of nanoparticles 54nm. The plant extraction and nanoparticle possess a significant alpha amylase inhibitory effect. Assay of alpha amylase inhibitory activity of plant extraction and plant extraction with yogurt was revealed the showed significant result in 100µg/ml and 90µg/ml of alpha amylase inhibition. Alpha amylase inhibition silver nanoparticles and nanoparticles with yogurt which showed the significant result in 100µg/ml and 10µg/ml. This study revealed that the silver nanoparticles of plant extraction have emerged as a promising alpha amylase inhibition to control hyperglycemia. However, in vivo animal model and cytotoxicity studies are required to explore the AgNPs.

No conflict of interest.

Authors are thankful and acknowledge the PG Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar-388121, Anand, Gujarat, India, for providing necessary support for research and laboratory facility.

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