Extraction of bioactive compounds and silver nanoparticle synthesis from fruits of Rauvolfia tetraphylla

doi:10.21203/rs.3.rs-4981754/v1

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Extraction of bioactive compounds and silver nanoparticle synthesis from fruits of Rauvolfia tetraphylla

https://doi.org/10.21203/rs.3.rs-4981754/v1

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Plant-derived bioactive chemicals have natural pharmacological effects. Rauvolfia tetrapylla has been used in the ancient days to cure a variety of ailments, including snake bites. The aim of the study is to scrutinize the bioactive chemicals isolated from Rauvolfia tetrapylla fruits in order to determine their antibacterial properties and to synthesis silver nanoparticles from the fruit extract. After 72 hours of incubation, a methanolic extract of the fruit was produced. Methanolic extract of the fruit synthesized silver nanoparticles. The fruit extract exhibited antibacterial activity against S. typhi and S. dysentriae. According to GC-MS report, there are 14 different chemicals identified in the fruit extract.

Rauvolfia tetrapylla

bioactive compounds

silver nanoparticles

antibacterial activity

Bacterial infections have always been treated using local plants and their concoctions. The role of herbal medicines [1] has the same potentiality as of the commercial antibiotics and moreover compare to antibiotics these plants has the capacity to overcome the resistance generated by the pathogens. Medicinal plants has enormous number of secondary metabolites which have the potential to inhibit the multiplication and growth of pathogens. Such an important medicinal plants are treated as weeds and garbages has been screened by recent scientists for the potential usage to fight against the microbes and act as a good source of antimicrobial agents. In this study a plant from the family Apocynaceae was chosen. The plant used was Rauvolfia tetraphylla which is a bush or a small tree found very commonly in tropical areas especially those in India, Mexico etc. Herbal medicines have been long used to treat diseases due to their pharmacological properties and Rauvolfia tetraphylla is no exception. There have been many studies done on this plant and on its phytochemical analysis [2]. This plant does have some good pharmacological properties. Different extracts of different plant parts might yield different compounds. This plant has been used by locals to treat several skin infection, helminthiasis, malaria and even for bite from snakes. It also has a variety of phytochemicals which may be the reason for its part in medicines. It has also exhibited some anti-inflammatory activity too as well as presence of antioxidant activities which may aid in treating cancer based on future researches [3]. Silver nanoparticles synthesized from plant extracts show good antibacterial activity. Stable nanoparticles produced from plant extracts have been an important research perspective [4].

Methanol was used to extract compounds from Rauvolfia tetraphylla and were used to do antibacterial assay against Salmonella typhi and Shigella dysenteriae. GCMS was done to know the compounds present in the extracts. Silver nanoparticles was synthesized from the methanolic extracts of the fruit of Rauvolfia tetraphylla and analysed using FTIR and SEM.

2.1. Collection site

The fruits of Rauvolfia tetraphylla were collected in and around the CLTRI campus, Chengalpattu. They were shade dried for 7 days to obtain dry fruit. The dried fruit was grinded into a fine powder in the lab using a mixer.

2.2. Preparation of crude extract

To 40g of the dried fruit powder was weighed was collected in a screw cap bottle. The solvent used was methanol in the ratio of 1:2 as in 40g of fruit powder and 80 ml of methanol. The mixture of the solvent and powder was kept in room temperature for 72 hrs. Then this extract was filtered with a muslin cloth and it was stored.

2.3. Phytochemical analysis

Standardized test procedures were executed from the standard references to detect the presence of secondary metabolites in the obtained extract [5].

2.4. Estimation of protein

The protein content was quantified using Lowry’s method as per the protocol Bovine serum albumin (BSA) solution was considered to be the standard and used in different concentrations of 0.2, 0.4, 0.6, 0.8 and 1.0mg respectively in 5 test tubes. The unknown extract sample was taken in different concentrations such as 0.5, 1.0 and 1.5 in 3 different test tubes and one blank. Here folin’s reagent was used as an indicator. The OD value was taken at 540nm in colorimeter [6].

2.5. In-vitro anti-bacterial activity

Rauvolfia tetraphylla (L) extract in-vitro antibacterial activity was done using sterile Mueller Hinton agar plates by agar well diffusion method [7]. The media was distributed onto sterile petri plates and allowed to solidify. The pure bacterial culture of Salmonalla typhi and Shigella dysenteriae were purchased from the Chettinadu hospital and spread on the two different plate’s separately using sterile L rod. Standard Whatman filter paper disks were immersed into the plant extract at different concentration such as 50, 100, 150 and 200µl with the help of sterile forceps and incubated at 37°C. The standard antibiotics used were ciprofloxacin for both Salmonella typhi and Shigella dysenteriae and the zone of inhibition was observed by measuring the diameter of clearance around the well.

2.6. GC-MS analysis

A shimadzu GCMS QP- pro was whipped with direct injector liner of 1mm and the split ratio was set to be 12:2. The oven temperature was at 100°C which was held for 2mins and ramped up to 180°C without holding time and then rate was increased at 3°C/ min till 280°C for 5 mins. The carrier gas (He) was set to 1ml/min of constant rate flow. The data peaks from mass spectrum was compared with the standard NIST library [8].

2.7. Synthesis of silver nanoparticles

To 0.15g of silver nitrate was dissolved along with 5ml of extract and kept in shaking incubator for overnight incubation. Change in color from green to dark brown color was used as a confirmation test for the silver nanoparticles synthesization [9]. The obtained sample of 2ml of the solution was transferred in each tubes and it was centrifuged for 13000rpm about 30 mins. The supernatant was discarded and to the pellet few ml of ethyl acetate was added and vortexed for 2 mins. The contents in both the tubes were combined to obtain a significant amount of pellet for further analysis [10].

2.8. UV-visible spectroscopy

Change in color of the filtrate with 1mM of silver resolution optically noticed over an ending of moment of truth displays the bio-decline of silvery ions to bright nanoparticles. The white nanoparticles made in the filtrate were listened by savoring of aliquots (1 ml) at various period breaks. Absorption calculation were carried out on UV-Visible spectrophotometer (CARY 100 BIO UV-Vis Spectrophotometer; Varian Inc., USA) at a finding of 1nm middle from two points 200 and 800nm ranges [11].

2.9. SEM analysis

The obtained bright nanoparticle sample was centrifuged for 20mins at 10,000rpm and drop coated thin mirror film. SEM study was performed utilizing the flipping through power microscope (JSM-6360, JEOL). Compositional reasoning and the confirmational of the presence of silver nanoparticle was confirmed through Energy Dispersive X-ray Spectroscopy (EDS) utilizing the SEM outfitted accompanying an EDAX attachment (carl zeiss, Germany) [12].

2.10. FT-IR analysis

The sample was injected into the infrared (IR) beam where the transmitted energy is measured and a spectrum was generated to identify the functional groups present in the sample [13].

3.1. Phytochemical analysis

The phytochemical analysis revealed the presence of alkaloids, glycoside, phenol, saponin, steroid, terpenoids, quinones, flavonoids, anthocyanins. Table 1 describes the presence and absence of phytochemicals in the prepared extract of the plant and Fig. 1 shows the estimation of the protein by Lowry’s method.

Table 1

Phytochemical analysis
S. No	Phytochemicals	Result
1	Alkaloids	++
2	Carbohydrates	--
3	Glycoside	++
4	Tannin	--
5	Phenol	+++
6	Saponin	+
7	Steroid	++
8	Coumarins	--
9	Terpenoids	++
10	Quinones	+
11	Flavonoids	++
12	Anthocyanins	+

Table 2

Antibacterial assay – Zone of inhibitions
S. No.	Organisms	Bioactive compounds in different concentrations
S. No.	Organisms	50 µl	100µl	150µl	200µl	Control (ciprofloxaxin)
1	Shigella dysenteriae	10mm	15mm	17mm	20mm	21mm
2	Salmonella typhi	2mm	4mm	6mm	10mm	13mm

3.2. In-vitro anti-bacterial assay

The extract was tested against 2 pathogenic bacteria Shigella dysenteriae showed 10 mm zone of inhibition in 50µl, 15 mm, 17 mm, 20 mm in 100, 150 and 200 µl respectively [14]. 21 mm was observed in control disc ampicillin (Fig. 2). Salmonella typhi showed maximum zone of inhibition compared to Shigella dysenteriae at 10 mm was observed at 200µl concentration; the minimum 2 mm was observed in 50 µl (Table. 2 and Fig. 2).

3.3. GCMS ANALYSIS

GC-MS chromatogram (Fig. 3) shows peaks for the tested extract and revealed 14 compounds present in the extract. Figure 4 contains peak table of compounds obtained in GCMS analysis and

3.4. UV-Visible spectroscopy

Peak was spotted at 420nm in UV-Visible spectrophotometer. Synthesis of Silver nanoparticles (Ag-NP’s) was confirmed by the presence of colour change and the same was affirmed confirmation by obtaining peak at UV-Vis spectrophotometer.

3.5. FTIR analysis

The FT-IR peak values will be used to confirm the presence of different functional groups, indicating the compound availability in the sample depending upon the peak value (Fig. 6).

3.6. SEM analysis

SEM image is used to measure the synthesized silver nanoparticles which is from 66.5nm to 85.5nm in the size (Fig. 7).

Increasing antibiotic resistance in bacteria has been a menace to healthcare. This research was done to show that plant extracts do have immense antibacterial activity. The study was done on fruits of Rauvolfia tetraphylla because there have been much less studies done on the fruit of this plant. Antibacterial assay was done using the extract which showed good result against Salmonella typhi and Shigella dysenteriae. Thus the study conclude by stating the importance of the fruit of this plant and its pharmacological significance.

Ethical approval

Not applicable

Authors Contributions

Dr. Krishnasamy Loganathan contributed for Research methodology and experimental works

Mr. Aditya Mohan Bharadwaj performed the experiments

Dr. Saravanan drafted the manuscript

Ms. Sheena Mariam Thomas helped for various Spectroscopic studies.

Funding

The authors declare that no funds, grants, or other financial support were received during the findings of this manuscript.

Conflict of Interest

The authors declare that they have no conflict of interest.

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No competing interests reported.

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You are reading this latest preprint version

Extraction of bioactive compounds and silver nanoparticle synthesis from fruits of Rauvolfia tetraphylla

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Collection site

2.2. Preparation of crude extract

2.3. Phytochemical analysis

2.4. Estimation of protein

2.5. In-vitro anti-bacterial activity

2.6. GC-MS analysis

2.7. Synthesis of silver nanoparticles

2.8. UV-visible spectroscopy

2.9. SEM analysis

2.10. FT-IR analysis

3. Result and Discussion

3.1. Phytochemical analysis

3.2. In-vitro anti-bacterial assay

3.3. GCMS ANALYSIS

3.4. UV-Visible spectroscopy

3.5. FTIR analysis

3.6. SEM analysis

4. Conclusion

Declarations

References

Additional Declarations

Status:

Version 1