Impact of Aqueous Leaf Extract of Punica granatum and Synthesized Silver Nanoparticles against Streptozotocin Induced Diabetes in Rats

Abstract

Punica granatum leaves are known for various therapeutic properties, but lack proper documentation. The present study was undertaken to study the antidiabetic impact of aqueous leaf extract of Punica granatum and silver nanoparticles synthesized using aqueous leaf extract of Punica granatum against streptozotocin induced diabetes in rats. Aqueous leaf extract of Punica granatum was prepared, phytochemical screening of the extract revealed the presence of various phytochemicals—alkaloid, tannin, saponin, total phenol and flavonoids. The aqueous leaf extract was employed to synthesize silver nanoparticles Synthesized silver particles were characterized using different techniques such as UV-visible spectrophotometer (UV-Vis), Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM), dynamic light scattering analysis (DLS), and zeta potential analysis (ZP). The characterization revealed the nanoscale (size <= 100 nm) of the synthesized silver particles, thus called as nanoparticles. Both the aqueous leaf extract and the synthesized nanoparticles were administered in streptozotocin-induced-diabetic rats to assess their antidiabetic effect. The results revealed that both the aqueous leaf extract of Punica granatum and the synthesized silver nanoparticles had significant antidiabetic activity against streptozotocin induced diabetes in rats. It was further found that the silver nanoparticles had enhanced antidiabetic activity as compared to the extract alone.

1. Introduction

Plants have considerable nutritional value and have been a primary source of medicines throughout human history [1, 2]. During the past 30 years, up to 50% of all medications have been derived directly or indirectly from plant sources. 35,000–70,000 plant species have been tested for therapeutic purpose so far. Plants, particularly those having ethnopharmacological use, served as the major source of medicine for early drug development [3].

Herbs have traditionally been a key component in pharmaceutical preparations. As individuals work to maintain their health, herbal medications are growing in popularity throughout emerging nations. Large flora, however, are still awaiting research to confirm their therapeutic effects [4, 5]. The treatment of many ailments involves the use of various plant components, including leaves, bark, fruits, roots, and seeds. Plants can also provide natural antimicrobials [5].

Nettle, dandelion, chickweed, neem and Tulsi are just are just a few examples of plants that are frequently found surrounding human settlements and are utilized as herbal remedies [6]. In addition to sheep and other animals, non-human primates, monarch butterflies, and other creatures also use plant parts as medicines when they are unwell [7]. Plant samples found in ancient burial sites are one form of evidence suggesting that Paleolithic humans were familiar with herbal medicine. For instance, Shanidar IV, a 60,000-year-old Neanderthal burial site in northern Iraq has produced significant amounts of pollen from eight plant species, seven of which are currently utilized as herbal treatments [8]. The development of new paths for herbal drugs might result from the intersection of rich information from several traditional medical systems. Ayurvedic medicine, as described in the Atharva Veda, the Rig Veda, and the Sushruta Samhita, has been practiced for thousands of years and employs hundreds of pharmacologically active herbs and spices, including curcumin-containing turmeric. One of the oldest and most prevalent traditional medicinal systems is ayurveda. The history of this conventional medical system is still not fully explored [9, 10]. On the clay tablets found in ancient Sumeria, hundreds of medicinal herbs, including myrrh and opium, are recorded. More than 800 plant remedies, including aloe, cannabis, castor bean, garlic, juniper, and mandrake, are listed in the ancient Egyptian Ebers Papyrus [7].

Punica granatum (Pomegranate) is a small tree which measures between five and eight meters tall and mainly found in Iran, the Himalayas and Northern India [11], China, USA and throughout the Mediterranean region. All the plant parts of Punica granatum such as seed, juice, peel, leaf, flower, bark, and root possess interesting pharmacological and toxicological activities [12].

The pomegranate tree is a long-lived tree that may survive for more than 200 years. The leaves are glossy, and the flowers are red, white, big, or variegated, with tubular calyxes that eventually turn into fruit. The pomegranate fruit has a grenade form, a deep red, leathery skin, and a crown-shaped calyx. A tiny quantity of sour and crimson liquid surrounds the seeds, which are separated by a white membrane pericarp [13].

Pomegranate has been consumed by people from all over the world from ancient times (4000–3000 BC). Pomegranate blossoms are associated with life, permanence, well-being, femaleness, fertility, wisdom, immortality, and purity [14]. It has also been utilized as folk medicine in numerous cultures, dating back to at least 1550 BC in the Egyptian Eber Papyrus [15]. According to recent study, pomegranate contains chemical components such as tannins, alkaloids, organic acids, flavonoids. The chemical components of pomegranate vary depending on the section of the fruit, seed, leaf, or peel [16]. Numerous studies on the antioxidant, anticarcinogenic, and anti-inflammatory properties of pomegranate constituents have been published, with a focus on the treatment and prevention of cancer, cardiovascular disease, diabetes, dental conditions, erectile dysfunction, bacterial infections, antibiotic resistance, and UV radiation-induced skin damage. Infant brain ischemia, male infertility, Alzheimer’s illness, arthritis, and obesity are other possible uses [13].

Nanotechnology entered the picture during the past vicennial, when research into the therapeutic benefits of plant extracts was at its zenith. Nanotechnology is a science, engineering and technology that deals with particles at the nanoscale, or at or below 100 nm. On December 29, 1959, physicist Richard Feynman gave a discussion titled “There’s plenty of room at the bottom” at a conference of the American Physical Society at the California Institute of Technology. It was a long time before the word “Nanotechnology” was coined. His presentation focused on how scientists would be able to manage and control individual atoms and molecules. Professor Norio Taniguchi created the term nanotechnology later in his research [17, 18]. The notion of synthesis of metallic nanoparticles mediated by plant extracts obtained from plant components such as leaves, roots, fruit peels, flowers, and whole plant, etc., utilizing polar or non-polar extraction medium began to gain momentum in the previous decade [19, 20, 21, 22]. This concept gained traction and was widely investigated by workers. Several metals, including silver, zinc, copper, iron, and others, can be used to create plant-mediated nanoparticles. The synthesized nanoparticles must then be characterized for properties such as size, shape, and stability using a variety of techniques such as scanning electron microscopy (SEM), ultraviolet visible spectrophotometer (UV–Vis), Fourier transform infrared spectroscopy (FT-IR), dynamic light scattering analysis (DLS), and zeta potential analysis [23, 24]. Many workers have reported that the silver nanoparticles synthesized using plant extracts have enhanced alleviating effect as compared to extract alone on various parameters of mammalian model in laboratory conditions [25, 26, 27, 28].

Considering the above apprehensions, this work was undertaken to synthesize, and characterize silver nanoparticles synthesized using aqueous leaf extract of Punica granatum (AgPg_aq), and assessing its medicinal impact on mammalian model.

2. Methodology

2.1 Plant materials

Punica granatum fresh tender leaves were obtained in the Ranchi area of India’s Jharkhand state. The plant leaves were washed with deionized water, disinfected for 5 minutes with a 0.1% HgCl₂ solution, dried in the shade for 15 days, and ground to a fine powder [29]. 50 g of sieved powder was subject to extraction using Soxhlet device with 350 ml distilled water. The extraction was carried out without interruption for 4 days. The obtained extract was filtered and concentrated using a rotary flash evaporator set at 45°C. The pure extract was kept at room temperature in airtight containers for further studies [30].

2.2 Preliminary phytochemical screening

Preliminary phytochemical screening assays were performed on Punica granatum aqueous leaf PG_aq extract using previously published standards [31].

2.3 Quantitative phytochemical screening

2.3.1 Alkaloid determination

For alkaloid determination, 200 ml of 10% acetic acid in ethanol and the sample was weighed into a 250 ml beaker. The extract was concentrated to one-fourth of its original volume in a hot water bath after being filtered and let to stand in a covered beaker for four hours. Until the precipitation was finished, concentrated ammonium hydroxide was applied drop by drop to the extract. Until the precipitate settled, the entire solution was let to stand. The precipitate was collected, cleaned with dilute ammonium hydroxide, and filtered. After thorough drying, alkaloid residue was gathered, weighed, and its percentage was measured and expressed in mg/g of plant extracts [32].

2.3.2 Tannin determination

After making slight modifications, the analyses of the tannin concentration in the aqueous leaf extract of Punica granatum was carried out in accordance with the International Pharmacopoeia [33] and Mulani et al. [34]. A 1 L conical flask was filled with 25 ml of the extract, 25 ml of indigo solution, and 750 ml of distilled water, the solution was made to 1 L using 0.1 N aqueous solution of KMnO₄. The blue colored solution changed to green color. Standard indigo carmine solution preparation: 6 g indigo carmine was dissolved in 500 ml of distilled water by heating, after cooling, 50 ml of 95–97% sulfuric acid was added, the solution was diluted to 1 L and then filtered.

The tannin content was calculated as percentage and expressed as mg/g of plant extract.

$$Tanin\%=\frac{\left(V-V_0\right)\times 0.004157\times 250\times 100}{g\times 25}$$E1

Where V is the volume of 0.1 N aqueous solution of KMnO₄ exhausted in titration (ml), V₀ is volume of 0.1 N aqueous solution of KMnO₄ for titration of the blank sample (ml). 0.004157 is tannin equivalent in 1 ml of 0.1 N aqueous solution of KMnO₄; g is mass of the sample taken for the analysis (g); 250 is the volume of volumetric flask (ml).

2.3.3 Saponin determination

Obadoni and Ochuko’s [35] published work was used to calculate saponin concentration in the aqueous leaf extract of Punica granatum. In a conical flask, 20 g of each ground sample was placed, and 100 ml of 20% aqueous ethanol was added. The flask was then heated for 4 hours on a hot water bath with continual stirring at around 55°C. the mixture was then filtered, and the residue was extracted once again using 200 ml of 20% aqueous ethanol. On a hot water bath at around 90°C, the combined extract was reduced to 40 ml. the concentrate was put into a 250 ml separator funnel, 20 ml diethyl-ether was added, and vigorous shaking was performed. The aqueous layer was saved, but the ether layer was not. The purifying procedure was carried out once again. N-butanol (60 ml) was added. The mixed n-butanol extracts were rinsed with 10 ml of 5% aqueous sodium chloride in triplicate. In a water bath, the residual solution was heated. The samples were dried in an oven after evaporation, weighed, and the saponin concentration was determined as a percentage and represented as mg/g of plant extract.

2.3.4 Total phenol determination

Folin-Ciocalteu’s reagent technique was used to assess the quantity of total phenol content in aqueous leaf extracts of Punica granatum [36]. 0.5 ml of extract and 0.1 ml (0.5 N) Folin-Ciocalteu’s reagent were combined and incubated at room temperature for 15 minutes. The absorbance at 760 nm was measured after 2.5 mL of saturated sodium carbonate solution was added and incubated for 30 minutes at room temperature. A positive control was gallic acid. Total phenol levels are given in milligrams per gram of extracted components (mg/g).

2.3.5 Flavonoids determination

Flavonoids concentration was determined following previously published work [37]. At room temperature, 10 g of sample was extracted with 100 ml of 80% aqueous methanol. The whole solution was filtered using #42 Whatman filter paper (125 nm). The filtrate was then transferred to a crucible and evaporated until dry over a water bath, and the weight and percentage amount were computed and represented in mg/g of plant extracts.

2.4 Nanoparticle synthesis

Biomolecules found in plant extracts can convert metallic ions to nanoparticles in a single step. This method is rather simple and may be performed at ambient temperature and pressure. The use of plant extracts in the production of metallic nanoparticles is eco-friendly [38]. Plant extracts can be prepared using a variety of solvents (polar or non-polar). Secondary metabolites found in plant extracts, which include alkaloids, phenolic components, terpenoids, tannins, saponins and others are responsible for reduction of metallic ions [39, 40, 41].

Silver nanoparticles were synthesized in this work utilizing aqueous Punica granatum leaf extract.

2.5 Characterization of synthesized nanoparticles

Several characteristics, including shape, size, capping materials, stability, etc., must be assessed for the synthesized nanoparticles. In this work, synthesized silver nanoparticles were subjected to UV-visible spectrophotometer (UV–Vis), scanning electron microscope (SEM), Fourier transform infrared spectrophotometer (FT-IR), dynamic light scattering (DLS) and zeta-potential analysis.

2.5.1 UV-visible spectrophotometer analysis

The fundamental idea of a UV-visible spectrophotometer is measuring the quantity of light absorbed and scattered by a sample (extinction, defined as the total of absorbed and scattered light). In order to measure the UV/visible light beam’s intensity both before and after it passes through the sample, a sample is positioned between a light source and a photodetector. To estimate the wavelength dependent extinction spectrum of the samples, these results are compared at each wavelength. The data is often shown as an extinction function of wavelength. To make sure that unexpected characteristics from the buffer are not included in the extinction-spectrum of the samples, each spectrum is background corrected using a buffer blank [42].

UV–visible spectrophotometers are effective tools for recognizing, describing, and studying nanomaterials because the optical characteristics of silver nanoparticles are sensitive to size, shape, concentration, aggregation state and refractive index close to the surface [43].

In this study the reduction of silver ions was monitored by measuring UV–visible spectrum of the reaction medium 5 hours after diluting a small aliquot of the sample with distilled water. UV–visible spectral analysis was performed using Perkin, Lambda 25 UV–visible spectrophotometer (USA).

2.5.2 Scanning electron microscope (SEM) analysis

Analysis performed using a scanning electron microscope may be utilized to look at the surface morphology of generated nanoparticles. The powder of the nanoparticles is first obtained and then coated with a heavy metal layer to produce SEM pictures. Once in the SEM step, the coated nanoparticles are bombarded with electron beams, which results in release of secondary electrons that are subsequently captured by the sensors to create pictures. The wavelength of an electron beam is shorter than that of visible light, allowing for the creation of high-resolution images of nanoparticles [39, 44]. In this investigation, silver nanoparticles were characterized using SEM equipment JIOL JSM-6390 LV (Jeol, Japan), which has supplemental software that can determine particle average size. The nanoparticles synthesized using aqueous leaf extract of Punica granatum was analyzed for surface morphology such as size (using accessory software) and shape using machine mentioned above and electron micrograph was obtained.

2.5.3 Fourier transform infrared spectroscopy (FT-IR) analysis

Nanoparticles’ chemical surfaces are extremely sensitive to FT-IR. FT-IR spectra are collected to identify functional groups on the surface of nanoparticles. During the creation of metallic nanoparticles, negatively charged molecules cap the metallic ions. The FT-IR measurement can help identify (only) functional groups in compounds that cap the metallic ions [45]. Shimadzu IR-prestige-21 (Shimadzu Corpn., Japan) FT-IR equipment was utilized in this investigation in the diffuse reflectance mode with a resolution of 4 cm⁻¹ in the range of 400–4000 cm⁻¹ to analyze the functional groups that could be involved in nanoparticle production.

2.5.4 Dynamic light scattering (DLS) and zeta-potential (ZP) analysis

Dynamic light scattering (also known as static, Rayleigh, or multi-angle light scattering) is a method for directly measuring particle size [46]. When light collides with a tiny object (such as a particle or molecule), it changes direction. A DLS equipment operates by shining a laser beam on a sample. A fast photon detector detects fluctuations in scattered light from a sample, and using known scattering angles, the whole particle size distribution inside a sample may be calculated. DLS devices used in a wide range of applications, including emulsions, micelles, polymers, proteins, colloids, and nanoparticle analysis [30, 44].

Light scattering analysis of nanoparticles is an essential method for determining nanoparticle size in solution. It quantifies the light scattered from a laser traveling through a colloidal solution by analyzing the variation of scattered light intensity as a function of time. The results of the light scattering study show a size distribution by number, intensity, and volume [44]. In the present study the light scattering and ZP analysis of nanoparticles were carried on Malvern Nano ZS (U.K.).

2.6 Animal model

In the investigation, Albino Wistar rats (175–200 g) were utilized. The rats were kept under typical laboratory conditions, with an ambient temperature of 25 ± 2°C and a relative humidity of 50 ± 15%, and a 12-hour dark-light cycle. The animals were fed a commercial pellet diet and were given free access to water [30]. The experiment was carried out after taking approval from Ranchi University’s Animal Ethics Committee (Proceeding no. 46, page no. 137).

2.7 Acute toxicity studies

Following OECD standards 423 [47], the acute oral toxicity of both aqueous extract and synthesized nanoparticles was assessed. The test’s guiding idea is to employ the fewest number of animals feasible. The aqueous extract and nanoparticles were administered to separate groups of animals orally at one of the predetermined dosages. The material was examined using a step-by-step approach, with each stage including three animals of a single sex, usually females, because female rats are more sensitive to chemicals than their male counterparts, making it easier to identify any lingering toxicities. The female rats chosen were nulliparous and not pregnant. The next phase was chosen by the lack of substance-related mortality in the animals dosed in one step. The study employed 15 healthy adult albino rats weighing between 175 and 200 g and aged 8–12 weeks. Prior to treatment, the rats were fasted overnight. Food was not allowed for 3–4 hours after medication. Since nothing was known about the lethal level of the drug in rats, three animals were utilized in each phase; the beginning dose was chosen as a minimum (5 mg/kg body weight (BW)). The chemicals were given in a single dosage by oral gavage. Individual animals were watched after dosing at least once in the first 30 minutes, occasionally throughout the first hour, with special attention during the first 4 hours and daily thereafter, for a total of 14 days. However, the period of observation was not set, i.e., dosed animals were caged separately after 14 days of observation and monitored for another 14 days. No mortality was reported up to a dosage of 2000 mg/kg body weight of rats [48].

2.8 Induction of diabetes

Streptozotocin (STZ), dissolved in 0.1 M sodium citrate buffer (pH 4.5), was administered intraperitoneally (i.p.) to rats after they had fasted for 18 hours, at a dosage of 50–60 mg/kg body weight. Following STZ injection, animals were monitored for the first 24 hours to look for any signs of allergic reactions, behavioral abnormalities, or convulsions. To treat the hypoglycemia caused by STZ, animals were given 5% glucose solution. None of the STZ-treated animals showed any abnormal reactions. Blood glucose levels were measured 72 hours after STZ treatment. In order to conduct the study, only animals with glycosuria and blood glucose levels between 200 and 300 mg/dl were chosen. They were then split into groups as described in experimental design (2.9). STZ was not administered to the control group and rest all the groups started with STZ-induced-diabetic rats [48].

2.9 Experimental design

Group 1: normal control group—received 0.5 ml of regular saline orally every day for 21 days. Before the administration of normal saline on day 0 at 10:00 am, then on days 3, 7, 14 and 21 at the same time, blood glucose levels were measured.

Group 2: diabetic control group—received 0.5 ml of ordinary saline orally each day for 21 days. Animals were watched for any signs of altered behavior, hyperglycemia and convulsions. On day 0, before giving this groups normal saline, the blood glucose levels were measured at 10:00 am.

Group 3: extract test group—received daily oral doses of leaf extracts at a dosage of 200 mg/kg (BW) for 21 days. On day 0, before the extract was administered, the blood glucose levels were measured at 10:00 am. The blood glucose levels were then measured 3, 7, 14 and 21 days after medicine was administered. The animals were watched for any signs of hypoglycemia and convulsion.

Group 4: nanoparticle test group—received daily oral doses of leaf extracts at a dosage of 200 mg/kg (BW) for 21 days. On day 0, before the nanoparticles were administered, the blood glucose levels were measured at 10:00 am. The blood glucose levels were then measured 3, 7, 14 and 21 days after medicine was administered. The animals were watched for any signs of hypoglycemia and convulsion.

Group 5: glibenclamide standard group—received daily oral doses of glibenclamide at a dosage of 0.5 mg/kg body weight for 21 days. On day 0, before the medicine was administered, the blood glucose level was measured at 10:00 am. The blood glucose level was then measured 3, 7, 14, and 21 days after medicine was administered. The animals were watched for any signs of hypoglycemia and convulsion.

3. Results

3.1 Phytochemical screening

Preliminary phytochemical screening of Punica granatum aqueous leaf extract revealed the presence of alkaloids, tannins, saponins, phenols, and flavonoids. The aqueous extract of Punica granatum was subjected to quantitative phytochemical screening after preliminary phytochemical screening. Figure 1 depicts the findings of quantitative phytochemical screening. Flavonoids were found in the greatest concentration (118 ± 2.5 mg/g), followed by phenol (80.5 ± 3.49 mg/g), saponins (65.5 ± 1.25 mg/g), tannins (3.3 ± 0.8 mg/g), and alkaloids (1.5 ± 0.05 mg/g).

Figure 1.

The concentration of alkaloid, tannin, saponin, total phenol and flavonoids found in the aqueous leaf extract of Punica granatum.

3.2 Synthesis of silver nanoparticles

In the current study, a color change from initial yellow to dark brown was noticed as soon as the Punica granatum leaf extract was mixed with aqueous solution of silver nitrate (Figure 2).

Figure 2.

Test tubes A to F showing change in colour of mixture of aqueous solution of AgNO₃ and PG_aq. (A) Colour (pale yellow) just after mixing of AgNO₃ and PG_aq; B to E. gradual colour change; F. final colour obtained.

3.3 Characterization of silver nanoparticles

3.3.1 UV-visible spectrophotometer

The reaction mixture was subjected to UV-visible spectrophotometer (UV–vis) analysis once the preceding step’s reaction was finished. Figure 3 shows the results of UV-vis analysis of AgPg_aq. According to several research [22, 23, 24, 25, 26], a surface plasmon resonance (SPR) peak with a wavelength between 400 and 490 nm suggests that the particle under investigation is in the nano range (below 100 nm).

Figure 3.

Graphical result obtained by UV–visible spectrophotometric analysis of AgPg_aq

3.3.2 Scanning electron microscope (SEM) analysis

Scanning electron microscope analysis can be used to examine the surface morphology of produced nanoparticles. To assess the size and shape of the AgPg_aq, SEM analysis was employed. The nanoparticles were spherical, rhomboid, and cubical in shape and had diameter ranging from 88.0 to 120 nm and average size of the particles were 98.93 nm (Figure 4).

Figure 4.

SEM images of AgPg_aq.

3.3.3 Fourier Transform Infrared (FT-IR) spectroscopy

The results of an FT-IR investigation of silver nanoparticles made from aqueous Punica granatum leaf extracts are shown graphically in Figure 5. The spectra showed broad transmission peaks at 3633.69 cm⁻¹, which corresponds to hydrogen bonded hydroxyl group (O∙H and H stretch) of alcohols and phenols. The 2102.41 cm⁻¹ peak corresponds to ∙SCN. The 1500.62 cm⁻¹ corresponds to ∙C〓C∙ stretch, which represents alkenes, 1361.74 cm⁻¹ corresponds to sulphonates, and 937.40 cm⁻¹ corresponds to C〓N stretch that represents the aliphatic amines.

Figure 5.

Result of FT-IR analysis of AgPg_aq.

3.3.4 Dynamic light scattering (DLS) and zeta potential (ZP) analysis

The results of light scattering analysis demonstrate a size distribution by intensity, volume, and number [22, 45] as depicted by the results of DLS analysis of nanoparticles synthesized using aqueous leaf extract of Punica granatum in Figures 6–8.

Figure 6.

Result of light scattering analysis of aqueous leaf extract of Punica granatum showing size distribution by intensity.

Figure 7.

Result of light scattering analysis of aqueous leaf extract of Punica granatum showing size distribution by volume.

Figure 8.

Result of light scattering analysis of aqueous leaf extract of Punica granatum showing size distribution by number.

Figure 6 shows results of light scattering (intensity distribution) analysis of aqueous leaf extract of Punica granatum showing two peaks with intensities of 91.0% and 9.0% at 96.5 nm and 141.6 nm.

Two peaks at 85.6 nm and 24.76 nm were seen in the volume distribution graph (Figure 7), illustrating the particle volume distribution in different size bins. According to the results, all of the particles produced were nanosized, with 84.2% of them measuring 85.6 ± 14.60 nm and 15.8% measuring 24.76 ± 2.114 nm.

One significant peak with a number distribution of 30% can be seen at 40.32 nm on the number distribution graph (Figure 8). This suggests that at least 30% of synthesized nanoparticles were 40.32 ± 4.413 nm in size.

The results of zeta potential analysis of AgPg_aq have been graphically presented as Figure 9. The zeta potential value of synthesized silver nanoparticles was −11.6 mV, having a standard deviation of 4.67 mV.

Figure 9.

Result of zeta potential analysis of AgPg_aq.

A particle’s zeta potential can range from +100 mV to −100 mV. The zeta potential study of Punica granatum aqueous leaf extract-produced silver nanoparticles reveal a peak of −11.6 4.7 mV with 100% area dispersion (Figure 9).

According to the results obtained from all the various techniques employed in this study to characterize the synthesized silver particles (as described earlier in the text), the size of synthesized silver particle exhibited significant stability with size in nano-range.

3.4 Impact of Pg aqueous leaf extract and silver nanoparticles synthesized using aqueous leaf extract of Pg on mammalian model

The results of impact of PG_aq and AgPg_aq has been graphically presented as Figure 10. The results are expressed as mean ± SD (n = 3). The results clearly confirm the antidiabetic activity of PG_aq and AgPg_aq. They were able to counter the diabetes induced by streptozotocin. The results of study of antidiabetic impact of PG_aq and AgPg_aq against streptozotocin induced diabetes in rats have been tabulated under Table 1. The data obtained was statistically analyzed using t-test and value of P < 0.05 was judged as significant. The table clearly shows that the group which received aqueous leaf extract of Punica granatum orally showed significant (P = 0.0002) decrease in plasma glucose levels from 45.9 ± 1.8 mmol/L (day 0) to 15.68 ± 1.3 mmol/L (day 21); in the other group which received silver nanoparticles loaded with aqueous leaf extracts of Punica granatum, the plasma glucose level decreased significantly (P = 0.0002) from 44.3 ± 2.0 mmol/L to 10.22 ± 1.8 mmol/L. Both the leaf extracts of Punica granatum and nanoparticles synthesized using leaf extract of Punica granatum showed significant antidiabetic activity against streptozotocin induced diabetes in rats.

Figure 10.

Comparative representation of impact of PG_aq and AgPg_aq against induced diabetes in rats (mean ± SD, n = 3).

Groups	Plasma glucose levels (mmol/L)
	Day 0	Day 3	Day 7	Day 14	Day 21
Group 1: normal control group	5.6 ± 0.52	5.66 ± 0.62	5.59 ± 0.66	5.46 ± 0.45	5.7 ± 0.51
Group 2: diabetic control group	45.5 ± 1.22	48.6 ± 1.5	49.33 ± 1.6	50.01 ± 1.52	50.11 ± 1.3
Group 3: PGaq (200 mg/kg)	45.9 ± 1.8	40.23 ± 1.75	28.65 ± 1.42	20.12 ± 1.4	15.68 ± 1.3*
Group 4: AgPgaq (200 mg/kg)	44.3 ± 2.0	39.43 ± 1.8A	21.95 ± 1.9B	18.33 ± 1.35C	8.22 ± 1.8^D,#
Group 5: Glibenclamide (0.6 mg/kg)	50.11 ± 2.3	18.26 ± 1.8	15.22 ± 2.0	10.43 ± 2.5	6.35 ± 1.8##

Table 1.

Showing the antidiabetic impact of PG_aq and AgPg_aq on diabetes induced by streptozotocin in rats (mean ± SD, n = 3).

*

Difference in plasma glucose levels on day-21 as compared to day-0: P = 0.0002.

^#

Difference in plasma glucose levels on day-21 as compared to day-0: P = 0.0002.

^##

Difference in plasma glucose levels on day-21 as compared to day-0: P = 0.0001.

^A

Difference in plasma glucose level of group 3 and group 4 on day 3: P = 0.7659.

^B

Difference in plasma glucose level of group 3 and group 4 on day 7: P = 0.0476.

^C

Difference in plasma glucose level of group 3 and group 4 on day 14: P = 0.409.

^D

Difference in plasma glucose level of group 3 and group 4 on day 21: P = 0.0283.

Statistical comparison of the antidiabetic activity of PG_aq and AgPg_aq showed that on there was non-significant (P = 0.7659) difference between plasma glucose levels on day-3 between rats receiving PG_aq (40.23 ± 1.75 mmol/L) and AgPg_aq (39.43 ± 1.8 mmol/L).

On Day 7 there was as significant decrease (P = 0.0476) in blood glucose level of group 4 (receiving AgPg_aq) (21.95 ± 1.9 mmol/L) as compared to group 3 (receiving PG_aq) (28.65 ± 1.42 mmol/L). On day 14 there was non-significant (0.409) difference between the plasma glucose levels of group 3 and group 4. On the 21st day group 4 exhibited significantly lower (P = 0.0283) plasma glucose level (8.22 ± 1.8 mmol/L) as compared to group 3 (15.68 ± 1.3 mmol/L). Group 5 which received the drug Glibenclamide always showed significant decrease in the plasma glucose level as compared to group 3 and group 4.

4. Discussion

4.1 Phytochemical screening

The amount and kind of phytochemical in each class are crucial to their effects. Flavonoids and phenols are substances found in broad variety of vascular plants, with over 8000 identified unique compounds [49]. They serve as antioxidants, antimicrobials, photoreceptors, visual attractants, feeding repellants, and light screens in plants [50]. Many studies have shown that both flavonoids and phenols have biological properties such as antioxidant, antiallergic, antiviral, and anti-inflammatory activity. However, the antioxidant activity of flavonoids and phenols have received the greatest attention due to its capacity to inhibit free radical production and scavenge free radicals [51]. The flavonoid content was found to be highest followed by phenols in the aqueous leaf extract of Punica granatum in the current investigation, indicating the extract to have significant antioxidant property. Dehshahri et al. reported the leaf extract of Moring peregrina to possess substantial antioxidant activities, which they attributed to the flavonoids and phenols present in the extract [52]. Shang-Tse et al. investigated the antioxidant activities of 18 indigenous tree species in Taiwan and discovered that flavonoid as well as phenols were key elements in the majority of the plants that had high antioxidant activity [53]. Wairata et al. in their correlation study found that there is a positive relationship of total phenolic and flavonoid contents with antidiabetic activity [54]. Praparatana et al. [55] and Iqbal et al. [56] in their studies on extracts of different plants have found straight correlation between the antidiabetic activities and the phenol and flavonoid contents of the studied plants.

Saponins cut blood lipids, reduce cancer risks, and improve blood glucose responsiveness. A high saponin diet can be used to prevent dental cavities and platelet aggregation, cure hypercalciuria in humans, and act as an antidote to acute lead poisoning [57]. Saponins, according to Barky et al. [58] and Metwally et al. [59] have been proven to lower blood glucose levels by rejuvenating insulin function, increasing plasma insulin levels, and releasing insulin from the pancreas. Saponin can also reduce glucose synthesis in the circulation [60]. Various other workers such as Singh et al. [61], Elekofehinti [62], and Jiang et al. [63] have reported that the saponins possess anti-diabetic properties.

Tannins have also been shown to have a variety of physiological effects, including anti-diabetic property and ability to speed up blood clotting, lower blood pressure, and lower serum cholesterol levels [64]. In their review, Laddha and Kulkarni noted that tannins play a role in the prevention and therapy of different diabetes problems [65]. Mohammed et al. [66] in their review found that the plant extracts having tannin as a constituent exhibited antidiabetic properties. Saratale et al. [67] studied the tannin extracted from grape pomace and reported it to possess antimicrobial, antioxidant and antidiabetic property as well.

Alkaloids are basic forms of salts with acids. They are nitrogen containing heterocyclic organic compounds of plant origin with different pharmacological activities [68]. Muhammad et al. in their review noted that alkaloids have antidiabetic activities [69]. Rasouli et al. concluded that the alkaloids have potentials to be used as alternative additives for treatment of hyperglycemia, suggesting need of further studies and researches [70]. Agrawal et al. studied the impact of alkaloids of Aerva lanata roots on streptozotocin-nicotinamide induced diabetes in rats and concluded that the alkaloids showed significant effects on streptozotocin-nicotinamide induced diabetes in rats [71].

Since all the phytochemicals (flavonoids, saponins, tannins, alkaloids) reported in the aqueous leaf extracts of Punica granatum have previously been reported to possess anti diabetic property, the leaf extract is expected to posses antidiabetic properties.

4.2 Synthesis of silver nanoparticles

Biomolecules found in plant extracts can convert metallic ions to nanoparticles in a single step. This method is rather simple and may be performed at ambient temperature and pressure. It is ecologically benign to employ plant extracts in the production of metallic nanoparticles [44]. In 2017, we introduced the name green nanoparticles for nanoparticles synthesized utilizing extracts from green plants’ leaves [22]. The term ‘green’ used here has a dual connotation. On the one hand, the phrase ‘green’ refers to a safer, cleaner, and more environmentally friendly approach of nanoparticle production utilizing plant extracts; on the other hand, the term ‘green’ refers to the presence of the green pigment chlorophyll in leaf extracts. As a result, any nanoparticle synthesized using extracts from chlorophyllous extracts may be referred to as “green nanoparticles,” whereas nanoparticles synthesized using extracts from achlorophyllous extracts may be referred to as “white nanoparticles” [44].

This is widely accepted confirmation for the reduction of silver ions by constituents of various phytochemicals present in the plant extract [19, 20, 22, 23, 24, 25, 44]. After 90 minutes, no color change was noticed, indicating that the reduction of silver ions by the phytochemicals included in the leaf extract was complete.

4.3 Characterization of silver nanoparticles

4.3.1 UV–visible spectrophotometer analysis

UV–visible spectrophotometry is a method for calculating extinction, which is total of light that has been absorbed and scattered by a sample [45]. Extinction is a measurement of how much light has been absorbed and scattered by a material.

In this study, silver nanoparticles synthesized from Punica granatum aqueous leaf extracts showed an SPR peak (Figure 3) at 420 nm (and 255 nm). Several investigations making use of Tridax procumbens [72], Argemone mexican [73], Papaya fruit extract [74], and Trianthema decandra [75] have observed absorbance peaks from 450 to 500 nm for silver nanoparticle formation.

4.3.2 Scanning electron microscope analysis

The size and shape of synthesized nanoparticles varies with the plant and metal characteristics. Alkammash [76] performed synthesis and SEM characterization of silver nanoparticles synthesized using Calotropis procera plant extracts, they reported the silver nanoparticles were spherical (size 8–20 nm). Our results are similar to those reported by Sharma et al. [77], Liaqat et al. [78], and Ali et al. [79].

4.3.3 Fourier transform infrared (FT-IR) spectroscopy

FT-IR analysis is highly sensitive to the chemical surface of nanoparticles. FT-IR spectra are obtained to detect the functional groups present on the surface of nanoparticles. During the creation of metallic nanoparticles, negatively charged molecules cap the metallic ions. Only the functional groups of substances that cap the metallic ion, may be identified via FT-IR analysis [45]. The FT-IR analysis’s measured spectra were compared to Coates’s previously reported reference values [80].

Multiple peaks were found by Rautela et al. [81] illustrating the complexity of Tectona grandis aqueous seed extracts. They noticed a change in the silver nanoparticle peak shapes and hypothesized that the functional groups in the seed extract were involved in the creation of the silver nanoparticles in their research [81]. Our results are in accordance with that of Rautela et al. [81].

4.3.4 Dynamic light scattering (DLS) and zeta potential (ZP) analysis

Particle size is directly measured by dynamic light scattering [46]. When light strikes a microscopic object or molecule, light scattering takes place. Applications for DLS devices include the study of emulsions, micelles, polymers, proteins, colloids, and nanoparticles [45]. A crucial method for figuring out the size of nanoparticles in a solution is DLS. To measure the light scattered from a laser passing through the colloidal solution, it studies the variation of scattered light intensity as a function of time.

The intensity data illustrates how light is scattered by a certain particle size bin; in this case, the data reveals that particles with a size of 96.5 ± 4.843 nm dispersed 91% of light. Particles that were 141.6 ± 32.40 nm in size dispersed 9% of the light. Thus, the graphs support the existence of silver particles synthesized at the nanoscale. Our results are in accordance with those of Ranjan et al. [23], Kumar et al. [44], and Kumar et al. [45].

Figure 7 represents the volume distribution graph of DLS analysis. The volume distribution graph shows the total volume of particles in different size bins. Our results were in accordance with the studies of Dandapat et al. [24], Rautela et al. [81].

Figure 8 represents graph showing size distribution of synthesized nanoparticles by number. The graph depicts that at least 30% of synthesized nanoparticles had size of 40.32 ± 4.413 nm. Our results are in accordance with Carvalho et al. [82].

4.3.5 Zeta potential analysis

A method for figuring out the surface charge of nanoparticles in a colloid (solution) is zeta potential analysis. A thin layer of ions with an opposing charge are drawn to the surface of nanoparticles due to their surface charge. As the nanoparticle diffuses through the fluid, a double layer of ions follow. The zeta potential of the particle refers to the electric potential at the double layer’s border. Most zeta potentials with values larger than +25 mV and less than −25 mV are highly stable. Our results are in accordance with previously published works [22, 45, 48].

4.4 Impact of Pg aqueous leaf extract and silver nanoparticles synthesized using aqueous leaf extract of Pg on mammalian model

Punica granatum is a fruit-bearing tree from Punicaceae family. It has healing qualities of quite an interest, which is due to the presence of phytochemicals alkaloids, tannins, saponins, phenols, and flavonoids [83]. Traditional systems of medicine, such as Ayurveda, Traditional Chinese Medicine, and Unani have cited various medicinal uses of leaves of Punica granatum [84, 85]. Fakudze et al. in their review discovered that Punica granatum has anticancer, antioxidant, anti-inflammatory, antiviral properties [83]. Das and Barman in their study reported that ethanolic leaf extract of Punica granatum showed antidiabetic property against rats with induced diabetes [86]. Streptozotocin is widely used to induce diabetes in animal models, such as rats and mice. It causes necrosis of β cells followed by β-cell loss and atrophy of islets [59].

Scientists have been more interested in metallic nanoparticles because of the induction of green synthetic processes in the synthesis of these particles, which are mediated by plant extracts, have decreased cost consequences, and repeatability and rapid reproducibility [45]. The metallic nanoparticles due to their small size, high surface area to volume ratio and other characteristics have attracted great interest of the scientific community. Silver nanoparticle synthesized from plant extract has been reported by many workers to have enhanced medicinal impact as compared to the extract alone [30, 44, 45]. Silver nanoparticles synthesized from plant extracts are known to show antimicrobial and anti-inflammatory activity, antibacterial, antiviral, and antifungal activities [45].

To the best of our knowledge, no studies on the effects of silver nanoparticles and their comparison with extract have been previously documented.

Owing to the above apprehensions both the aqueous leaf extract of Punica granatum (PG_aq) and nanoparticles synthesized using aqueous leaf extract of Punica granatum (AgPg_aq) were administered in rats (as described in the ‘materials and methods’ section), in which diabetes was induced using streptozotocin. The results revealed that the aqueous leaf extract of Punica granatum and the synthesized nanoparticles have significantly reduced the increased blood sugar induced by streptozotocin toxicity. Statistical analysis revealed that the silver nanoparticles were more effective as an anti-diabetic agent as compared to the extract alone. Similar observations were noted in many experimental diabetes researches [71, 72, 73, 74]. Our results are in accordance with the findings of Balan et al. [87], Wahab et al. [88], and Bhagyalakshmi et al. [89].

Thus, on the basis of results of present study it can be concluded that the aqueous leaf extract of Punica granatum and silver nanoparticles synthesized using aqueous leaf extract of Punica granatum showed significant antidiabetic activity on streptozotocin diabetic rats. Further it was found that the AgPg_aq had significantly higher alleviating effect against streptozotocin diabetes in rats as compared to the impact of the aqueous extract alone.

5. Conclusion

Thus, based on results of present study it can be concluded that the aqueous leaf extract of Punica granatum and silver nanoparticles synthesized using aqueous leaf extract of Punica granatum showed significant antidiabetic activity on streptozotocin diabetic rats. The aqueous leaf extract loaded silver nanoparticles had enhanced antidiabetic impact as compared to the extract alone which is a novel finding.

Acknowledgments

The authors declare that this work has not received funding in any form from any agency/institution. The authors acknowledge the laboratory facilities extended by the Head of the Department of Zoology, Ranchi University and Central Instrumentation facility, Birla Institute of Technology, Ranchi.

Conflict of interest

The authors declare no conflict of interest.