Phytochemical Profiling and Green Synthesis of Zinc Oxide Nanoparticles using Aqueous Extracts of Cynanchumviminale(L.)L. (Apocynaceae)

Jinu Mathew¹ , Megha Mariet¹ , Sruthy B² , Krishnaraj M V³

^1*Department of Chemistry and Centre for Research, Baselius College, Kottayam, Kerala, India

²Accuratus Scientia Natural Product Research and Development Pvt.Ltd, Attingal, Kerala, India

³Department of Botany, Baselius College, Kottayam, Kerala, India

Corresponding Author Email: jinumathew@baselius.ac.in

DOI : https://doi.org/10.51470/ABP.2025.04.03.35

Abstract

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1.Introduction

Plants are extensively used as home remedies and play a significant role in traditional treatments. The WHO supports the use of traditional medicine, and it has been proven to be efficacious and safe. Plants often create these compounds to defend themselves, but new studies showthey can shield people from illness[1]. Understanding these chemical components of plants is important for developing therapeutic agents and identifying new sources of profitable phytocompounds for synthesising complex chemicals and determining the true meaning of traditional medicines. Numerous biochemicals found in plant extracts, including alkaloids, polysaccharides, vitamins, amino acids, proteins, enzymes, tannins, phenolic acids, saponins, and terpenoids, have been identified in studies as potential contributors to the green synthesis of nanoparticles[2–6]. These phytochemicals impart significant medicinal and biological activities to nanoparticles.

Cynanchumviminale(L.)L. isa xerophytic leafless plant of the Apocynaceae family, the fifth most valuable medicinal plant family among Angiosperms[7]. Locally, it is known as ‘Somalatha’. Traditionally, the plant is used to prepare “somarasas” (rejuvenating drink) used by Aryans[8]. Previous literature showed that this plant has been used by ethnic communities in India[8]. Recent studies give evidence that aqueous extract of the stem of the Cynanchumviminale(L.)L.shows pharmacological effects such as anxiolytic, antipsychotic, anti-inflammatory, hepatoprotective, and CNS inhibitory [9].

The plant’s medicinal properties are due to its phytochemical compounds, which affect the mind and body. As a result, phytochemical screening might discover a variety of significant substances that could be employed as the basis of current medications for treating various ailments. spontaneous reducing and capping capacities have attracted significant attention towards green nanomedicine. CuO NPs synthesised from the stem extract of Sarcostemmaacidumwere found to have better anti-inflammatory activity[10].

Silver nanoparticles (SNPs) synthesised by xerophytic plants, Cynanchumviminale and Cynanchumsarcomedium [10] exhibited a maximum absorbance at 500 nm in the UV-Vis spectrum for C. viminale synthesised SNPs.SEM revealed thatC. Viminale produced agglomerated nanoparticles with an average diameter of about 60–68 nm.Additionally,C.sarcomediumextract was used to synthesize spherical nanoparticles with an average size of roughly 60-85 nm. SNPs produced by both lants showed a strong silver signal at 3keV according to EDAX analysis.

 Studies on the synthesis of copper oxide nanoparticles in the30-70nm diameter range and polymer functionalized copper oxide nanoparticles in the diameter range of 27-32nm were also reported. The reports revealed maximum in vitro antidiabetic activity of polymerised copper nanoparticles compared to biosynthetic copper nanoparticles from Cynanchumviminale stem extract[11].

The present research aims to quantify some of the important phytochemicals present in the aqueous extract of the aerial part of Cynanchumviminale(L.)L.(somalatha) ,which the rural people widely use to treat various diseases and to study the effect of phytochemicals on the synthesis of zinc oxide nanoparticles.

1.Materials and methods

1.1. Plant collection

The aerial part of Cynanchumviminale(L.)L. were collected in August 2023 from the Thiruvananthapuram district of Kerala, India. The plant material was shade-dried, powdered, packed in closed containers, and stored for phytochemical studies.

1.2. Preparation of plant extract

30g of plant material was extracted using a hot extraction method using 900 mL water. It is boiled for 5-6 hours in a heating mantle to prepare aqueous extracts. Subsequently, it is filtered through a muslin cloth and preserved at around 4^OC for future utilization.

1. Phytochemical screening

1.3.1. Qualitative analysis of phytochemicals

The principal phytochemical components of aqueous extract were qualitatively tested using the conventional methods outlined by Harborne et al[12]. The primary metabolites analysed included carbohydrates, reducing sugars, starch, and proteins, while the secondary metabolites comprised alkaloids, phenolic compounds, saponins, and flavonoids.. 2.3.2.Quantitative analysis of phytochemicals

2.3.2.1. Total alkaloid content.

The total alkaloid content of the aqueous extract was determined spectrophotometrically using Atropine as a standard, as described by Selvakumar S with slight modification[13].1mL of the aqueous extract of the sample is evaporated to dryness. It is then dissolved in 2N HCl and filtered 1 mL into a separating funnel. Wash three times with 10mL chloroform. Shake thoroughly after adding 0.1N NaOH, 5ml of Bromocresol green and 5mL of phosphate buffer. Extract using 1,2,3,4 ml of chloroform while vigorously shaking. Collect in a10 mL volumetric flask and dilute with chloroform. The absorbance was measured at 470nm. Atropine was used to plot a calibration curve and the total alkaloid content was expressed as Atropine equivalents.

2.3.2.2. Total flavonoid content

Estimation of flavonoids was done using the AlCl₃ method, using Quercetin as a standard as described by Madhavi et.al. with slight modification[14].1mL of the plant extract and 4 mL of water were taken. After 5 minutes,0.3 mL of 5% sodium nitrite and 0.3 mL of 10% AlCl₃ were added to it. After 6 minutes of incubation at room temperature, 2mL 1M NaOH was added to the reaction mixture. It was made up to 10mL with distilled water. Absorbance was measured at 510nm against a blank. The calibration was drawn using Quercetin as a standard.

2.3.2.3 Total phenolic content

The total phenolic content of aqueous extract of the sample was determined spectrophotometrically according to the Folin-Ciocalteu method with modification[15]. Different concentrations of the aqueous extracts of the Cynanchumviminale(L.)L.was mixed with 0.4 ml Follin-Ciocalteu reagent (diluted 1:10v/v). 4 mL of sodium carbonate solution was added after five minutes. The final volume is made up to 10 ml with distilled water which is then let  to stand at room temperature for 90 minutes (about one and a half hours). Absorbance was measured at 750 nm. A calibration curve was created with catechol solution as a standard, and total phenolic content was expressed in mg catechol/ g of dry weight,using the standard graph.

2.4. Green synthesis of zinc oxide nanoparticles

To 25 ml of 0.5 M zinc acetate dihydrate,2.5 ml of plant extract was added. To continue maintaining the mixture’s pH at 8, 2 M NaOH was added gradually. The mixture was subsequently agitated at 70°C for 30 minutes to achieve full reduction and form a white precipitate. Powdered ZnO nanoparticles were produced by collecting the resulting material through decantation, washing it with distilled water to eliminate residues, and then oven-drying it overnight at 70 °C. For further analysis and uses, the dried sample was kept in an airtight jar at ambient temperature. The yield % was subsequentlydeterminedusing the following formula[16]:

Yield (%) = (Experimental weight of ZnO/Theoretical weight of ZnO) × 100

• Qualitative and quantitative analysis of phytochemicals in the aqueous extract of Cynanchumviminale(L.)L after the synthesis of ZnO Nanoparticles

2.5.1. Qualitative analysis of Phytochemicals

Major phytochemical constituents of aqueous extracts, like carbohydrates, reducing sugars, starch, protein, amino acid, alkaloids, phenolics, saponins and tannins, were qualitatively analyzed according standard protocols outlined by Harborne et al.

2.5.2. Quantitative analysis of phytochemicals

2.5.2.2. Total flavonoid content

Estimation of flavonoids was done using AlCl₃ method, which used quercetin as a standard[14].1mL of the plant extract and 4 mL of water was taken. After 5 minutes,0.3 mL of 5% sodium nitrite and 0.3 mL of 10% AlCl₃ were added to it. After 6 minutes of incubation at room temperature, 2mL 1M NaOH was added to the reaction mixture. It was made up to 10mL with distilled water. Absorbance was measured at 510nm against a blank. The calibration was drawn using quercetin as a standard.

2.5.2.3. Total phenolic content

The total phenolic content of aqueous extracts of the sample was determined spectrophotometrically using the Folin-Ciocalteu method with slight modification[15]. Different concentrations of the aqueous extract of the Cynanchumviminale were mixed with 0.4 mL Follin Ciocalteu reagent (diluted 1:10v/v). After 5 minutes, 4 mL of sodium carbonate solution was added. The final volume is made up to 10 ml of distilled water and allowed to stand for 90 minutes (about one and a half hours) at room temperature. Absorbance was measured at 750 nm. A calibration curve was constructed using catechol solution as a standard, and total phenolic content was expressed in mg catechol/ g of dry weight using the standard graph.

2.6. Characterisation of ZnO NPs

Using a double-beam UV–visible spectrophotometer (T70/T80 series UV/Vis Spectrophotometer) in the 200–800 nm range, the synthesised ZnO NPs from the aqueous extracts of Cynanchumviminale were confirmed and characterised in order to see the distinctive peak verifying ZnO NPs formation. Using a scanning electron microscope (JEOL JSM5800) with an accelerated voltage of  5-20 kV the sample’s surface morphology was recorded.

3.Results and discussion

3.1. Phytochemical screening

3.1.1. Qualitative analysis of phytochemicals

Preliminary phytochemical screening of the aqueous extract revealed the presence of primary and secondary metabolites. The results are shown in Table 1.

3.1.2. Quantitative analysis of phytochemicals

Based on the qualitative analysis of phytochemicals, quantitative analysis was also done for major phytochemicals such as alkaloids, flavonoids, and phenolic compounds. Phenolics and flavonoids are known for their antioxidant properties, which enable them to neutralize harmful reactive oxygen species (ROS) within the plant cells. So, detecting phenolic and flavonoid compounds in plants is crucial to their potential to enhance our body’s antioxidant capacity, thereby offering cardioprotective, anti-cancer, anti-diabetic, anti-ageing, and neuroprotective benefits.

 

3.1.2.1 Total alkaloid content

Alkaloids are nitrogen-containing secondary metabolites used as medications to treat chronic diseases such as cancer, diabetes, and neurological disorders. Aqueous extract of Cynanchumviminale(L.)L.contains 26.21063 mg/g of the dried sample. It is expressed using atropine as a standard.

3.2. Green synthesis of ZnO Nanoparticles

This study developed a cost-effective, eco-friendly method for green ZnO NP synthesis from zinc acetate dihydrate using aqueous Cynanchumviminale extracts as reducing and capping agents. The aqueous extract is considered eco-friendly over organic extracts and found more appropriate for the green synthesis of ZnO NPs[17, 18]. Further, ZnO NPs are studied using instrumental techniques such as UV-Visible and SEM analysis. Several phytochemicals present in aqueous extracts of Cynanchumviminale, such as flavonoids, phenolics, and aromatic compounds, exhibit antioxidant properties, and consequently, can play an important role as reducing and/or capping agents in nanoparticle synthesis.

Quantitative analysis of important phytochemicals, such as flavonoids and phenolic compounds, which act as significant reducing and capping agents for nanoparticle synthesis, was performed. It was observed that phenolics and flavonoids in the plant extract can trigger the reduction of zinc acetate[19].

3.2.1Qualitative analysis of Phytochemicals

Phytochemical screening of the aqueous extracts of Cynanchumviminale was done after the synthesis of ZnO nanoparticles. The results were shown in Table 4

aqueous Cynanchumviminale extract after the synthesis of Nps.

Where; + Positive, ± Trace, – Negative

3.2.2. Quantitative analysis of Phytochemicals

3.2.2.1. Estimation of total phenolic content after the synthesis of ZnO nanoparticles

The total amount of phenolics as determined using the regression equation of calibration curve (y=0.0806x+0.0086, R²=0.9953) and expressed in catechol equivalent was 21.3920 mg/g of plant extract. The standard calibration curve of catechol is given in Figure 4. Table 5 shows the total phenolics in aqueous extracts of Cynanchumviminale determined by the Folins-Ciocalteau method.

 

 

 

3.2.2.2. Estimation of total flavonoid content after the synthesis of ZnO nanoparticles

 

The total flavonoid content was quantified using the calibration curve’s regression equation(y=0.3956x+0.168, R²=0.9975) and expressed in quercetin equivalent was 6.7324 mg/g of plant extract. The standard calibration curve of Quercetin is given in Figure 5. Table 6 shows the amount of total flavonoids in aqueous extracts of Cynanchumviminale determined by the AlCl₃ method after synthesis of ZnO nanoparticles.

 

 

3.3. Characterisation of ZnO NPs

 

3.3.1. UV–Visible spectral analysis

Secondary metabolites in plants convert zinc ions in solution into zinc oxide. The plant extract functions as both a reducing agent and a stabilizing agent. This was validated by UV-visible spectrum analysis in the region of 280nm-800nm. The produced ZnO NPs were dispersed in deionized water and subjected to sonication for 10 min to detect the UV–visible spectra. The UV-Vis spectra confirmed the successful formation of ZnO NPs using Cynanchumviminale aqueous extract, with a strong absorption peak at 360 nm(Fig. 6). For ZnO nanoparticles, the absorbance peak is reported between 310nm and 360 nm[20].

 

 

 

 

.3.3.2. Scanning Electron Microscope

 

SEM images of ZnO nanoparticles biosynthesised by C. viminale extract were obtained, but the precise shape of the nanoparticles could not be predicted. The particle size was 100nm(Fig.7). The hydrogen bonding and electrostatic attraction between bioorganic capping molecules and NPs have resulted in their aggregation[21].

 

 

 

 

 

     The elemental composition of ZnO NPs was obtained from EDX analysis. EDX analysis of ZnO NPs generated with C.viminale extract revealed a significant zinc signal at 1.26 keV, indicating the nanoparticles’ elemental composition and chemical purity. ZnO production is indicated by high Zn and O content. Stabilising agents derived from plant extract were detected in the EDX spectra, particularly carbon, nitrogen, and other elements.

 

 

 

Discussion

The phytochemical examination of C.viminale‘s aqueous extract revealed the presence of carbohydrates, reducing sugars, starch, protein, amino acids, alkaloids, phenolic compounds, and saponin. The quantitative analysis of three identified phytochemicals (alkaloids, flavonoids, and phenolics) revealed a high amount of phenolics (32.731 mg/g), followed by flavonoids (26.2106 mg/g) and alkaloids (9.2581 mg/g). For therapeutic purposes, phenolics are of utmost significance. Diaz et al. also reported that medicinal plants’ anti-inflammatory and antioxidant activities were correlated with the levels of phenolic and flavonoid compounds[22]. Tukun et al. reported that phenolic content is significantly connected to antioxidant activity[23]. Also, phenolic phytochemicals play various protective roles against abiotic stresses, such as UV light, and abiotic stresses, namely predator and pathogen attacks.

Flavonoids are polyphenolic chemicalsprotecting plants from UV radiation, pathogens, and oxidative stress. According to Okoli and Okere, flavonoids are powerful water-soluble super antioxidants and free radical scavengers that protect cells from oxidative damage, have significant anticancer properties, and suppress tumour development[24]. ROS are free radicals that serve as messengers inside cells, but when present in high concentrations, they can harm proteins, RNA, and DNA. Cells have developed numerous ways to fix the damage caused by these species, which is why antioxidants could be helpful as medicine. Due to their structure, flavonoids can function as reducing agents in various processes, providing an antioxidant effect. Flavonoids’ underlying methods include scavenging ROS, blocking oxidases that produce the superoxide anion, chelating trace metals, and activating antioxidant enzymes. The high phenolic and flavonoid content supports the traditional use of C.viminale as an anti-inflammatory agent.

Plants are the most popular green and easy method for creating nanoparticles because they provide the large-scale manufacturing of stable nanoparticles in various sizes and forms. Using natural plant extracts is economical, easy, and environmentally beneficial. Numerous studies have shown how to use various plant extracts to create zinc oxide nanoparticles in an environmentally friendly manner[17, 25–29]. However, the C.viminale plant has not been reportedly used for ZnO NPs synthesis. The green method synthesized zinc oxide NPs with a preferred size of 100nm. The fundamental mechanism for the green synthesis of ZnO nanoparticles remains incompletely elucidated. Free amino and carboxylic groups of proteins, alkaloids, phenolics, or flavonoids found in plant extracts may bind to zinc (Zn²⁺) surfaces, causing the production of ZnO NPs. Protein amides and the C=O, C=O–C, and C=C groups of heterocyclic compounds may have stabilising properties[30]. The flavonoid and phenolic compounds present in the extract have been used up for the synthesis of ZnO NPs, as evidenced by the quantitative determination of phenolics(21.320 mg/g) and flavonoids(6.7324mg/g) after the synthesis of NPs.

Future research will examine the use of ZnO NPs in various domains where their anti-cancer, anti-bacterial, anti-microbial, anti-tyrosinase, and anti-biofilm properties are employed; size and shape are crucial factors.

Conclusion

The therapeutic value of medicinal plants is determined by their phytoconstituents, either separately or in combination. In addition to its central nervous system inhibitory properties, Cynanchumviminale is utilised as an antioxidant, antibacterial, anti-inflammatory, antifertility, anti-ulcer, anxiolytic, and antipsychotic. The results show that the plant’s aqueous extracts contain carbohydrates, reducing sugars, starch, protein, amino acids, alkaloids, phenolic compounds, and saponins. Quantitative examination of three identified phytochemicals (alkaloids, flavonoids, and phenolics) revealed a high amount of phenolics (32.731 mg/g), followed by flavonoids (26.2106 mg/g) and alkaloids (9.2581 mg/g). Secondary metabolites in plants convert zinc ions in solution into zinc oxide. The plant extract acts as both a reducing and stabilising agent. The quantitative study of the aqueous extracts post-synthesis of ZnONps indicated a reduction in the levels of significant secondary metabolites, specifically phenolics (21.320 mg/g) and flavonoids (6.7324 mg/g). This finding suggests that they are depleted throughout the ZnONps production process. UV-visible spectral analysis verified this, revealing a 360 nm peak characteristic of ZnO nanoparticles. SEM examination validates the size and structure of ZnO, whose purity and composition were confirmed by EDX. This study suggests that an ecologically benign method for producing metallic nanoparticles will considerably aid in developing an important synergy between material science and biology. The physical properties of the produced nanoparticles were also proved, and the current work is also an efficient pilot research to manufacture ZnO nanoparticles using an environmentally benign method.

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