Evaluation of Anti-oxidant, Anti-microbial and Phytochemical analysis of different parts of C. bipinatus

 

Tejaswi Jasti1, Manisha Senapathi1, Sasank P1, Varaprasad Bobbarala1, Kandra Prameela2

1Department of Biotechnology, GIT, GITAM Deemed to be University, Visakhapatnam, India, 530045.

2Assistant Professor, Department of Biotechnology, GITAM Institute of Technology,

GITAM University. Visakhapatnam, India.

*Corresponding Author E-mail: pkandra@gitam.edu, chprameela5@gmail.com

 

ABSTRACT:

Plants are sources of many natural active compounds used to prevent several diseases.  The various parts of plants like leaf, stem, flower and root are rich of these bioactive compounds. From ancient times these natural compounds have been using for treatment and prevention of diseases. Cosmos bipinnatus is an herbaceous, medicinal plant. The present study was aimed with the objective to antioxidant, antimicrobial and phytochemical screening, total phenolic and total flavonoid content of different solvent extracts of different parts of Cosmos bipinnatus. Flavonoids were abundantly found in the leaves and the root of Cosmos bipinnatus. Steroids were absent only in the leaves and flowers of Cosmos bipinnatus while terpenoids were completely absent in the flowers of the plant. Alkaloids were absent in the leaves extracted from all the three solvents and root sample extracted using ethyl acetate and stem sample extracted using ethanol. Proteins are absent in all the parts of the plant extracted using all the three solvents namely ethanol, ethyl acetate and hexane. Tannins are absent in the entirely in the flower in addition to the stem sample taken using the solvent hexane. Saponins are present all the parts like leaf, stem and flower but are not found in the root extract of the plant. The total phenolic and flavonoid contents of the extracts were calculated by using the standard gallic acid and quercetin graphs to find out the concentration. The total phenolic content is high for the plant sample stem that is extracted by using hexane as the solvent the value is 790µg/mg. The total flavonoid content is high for the plant sample stem that is extracted by using hexane as the solvent the value is 150µg/mg. The antioxidant activity was also found out by using various assays like FRAP and DPPH where the percentage of inhibition was found out. The highest percentage of inhibition for leaf  was found out to be at 120µg/ml with the solvent hexane that is 88.86%. The highest percentage of inhibition for stem was found out to be at 60mg/ml using ethanol that is 89.92%. The highest percentage of inhibition for flower was found out to be at 60mg/ml by ethyl acetate that is 91.42%. The highest percentage of inhibition for root was found out to be at 60mg/ml that is 97%. From the FRAP results it is evident that the antioxidant activity is highest for the root sample.

 

KEYWORDS: Phytochemical analysis, Phenolic content, flavonoid, antioxidant, antimicrobial activity.

 

 


INTRODUCTION:

Plants produce a diverse range of secondary metabolites that have recently gained renewed interest for their antimicrobial and antioxidant properties. These may be produced constitutively or in response to external stress.

 

These compounds are advantageous because of their structural diversity and unique bioactivity which are more favourable than synthetic chemicals. Due to diversity in chemical nature of natural compounds from plant extracts and have opportunities to use as new drugs for treatment of diseases1. Discovering of novel drugs having biological activities is an continuous emerging need against infectious diseases2. However, most of the microorganisms are developing resistances against certain drugs and identification of new agents with antioxidant and antimicrobial activities is one of the challenging in recent years3. Although some of the synthetic drugs are available but plant derived drugs are to be considered as safe without or less side effects when compared to synthetic ones4,5. Many plants are rich sources of these antimicrobial agents and they are of secondary metabolites6-9. As infectious agents are getting resistance exploitation of natural sources and usage of plant extracted bioactive compounds is highly interesting in current years.

 

Cosmos bipinnatus, is famously known as the garden cosmos of Asteraceae family. It is an herbaceous, flowering and half-hardy yearly medium-sized medicinal plant. C. bipinnatus is self-sowing10. The plant ranges from 0.6 to 1.8 m in height with an open and sprawling habit11. It has varietal colours in full sun and flowering with partial shade. It is used to against stomach disorders pain and headaches and also against bed bugs and lice indicating its insecticidal property. Apart from ecology and morphological descriptions on this plant species, there is no scientific information to validate its medicinal potentials12. Wild populations of this plant rarely persist. It is having pharmacological activity and used as anti-inflammatory, jaundice, splenomegaly, antioxidant and also used as antimicrobial agents13. Only very few studies were reported on C. bipinnatus. Thus, the purpose of the study was to find qualitative phytochemical screening to ascertain the presence of bioactive metabolites such as tannins, alkaloids, triterpenoids, saponins and steroids and to estimate the total phenolic and flavonoid content of ethanol, hexane and ethyl acetate solvents extract of different parts root, leaf, stem, and flower to explore the underlying curative potentials of the plant in the form of antioxidant, antibacterial, antifungal activities.

 

MATERIALS AND METHODS:

Materials:

Chemical reagent DPPH was purchased from Sigma (Sigma-Aldrich India). Gallic acid standard solution (Loba Chemie, Mumbai). All other reagents used were of analytical grade.

 

Collection and identification of the plant:

The plant sample has been collected from Chityal mandal, Warangal district, Telangana state.The plant specimen was identified as Cosmos bipinnatus with the help of Professor Pandal, Department of Botany, Andhra University, Visakhapatnam. The sample was deposited in Botany Department Herbarium (AUV) Andhra University got voucher specimen number 23338. Different parts like root, stem, leaf and flower were separated, and shade dried for a period of 60 days and sample were grinded into the fine powder and stored in glass bottle at 4°C until further use.

 

Solvent extraction of different parts of C. bipinnatus:

The dried different parts of C. bipinnatus in the form of powder (1kg) has been taken and subjected to soxhlet solvent extraction process by taking 4L of ethyl alcohol, hexane and ethyl acetate for 24 hours. The crude extracts of C. bipinnatus thus obtained were stored in brown bottles and preserved in a freezer at -20şC until further use.

 

Preliminary phytochemical screening of C. bipinnatus:

The preliminary phytochemical screening tests for the presence of carbohydrates, proteins, tannins, phenols, saponins, flavonoids, steroids, glycosides, terpenoids and alkaloids were carried out for the ethanol, hexane and ethyl acetate crude extracts of Cosmos bipinnatus14.

 

Quantitative phytochemical analysis:

Determination of total phenolic content of C. bipinnatus:

The total phenolic content of each part of plant extract was determined by the Folin- Ciocalteau reagent method with some modifications15. All measurements were done in triplicates. The total phenol content was expressed in terms of Gallic acid equivalent (mg/g)16.

 

Evaluation of total flavonoid content of C. bipinnatus:

The total flavonoid content of crude extract was determined by the aluminium chloride colorimetric method. 50μL of crude extract were made up to 1mL with methanol, and all other chemicals according to the procedure were added and mixture was allowed to stand for 15 min, and absorbance was measured at 510nm. The total flavonoid content was calculated from a calibration curve, and the result was expressed as mg quercetin equivalent/g dry weight17.

 

Evaluation of antioxidant activity of C. bipinnatus

FRAP assay:

Ferric reductive Antioxidant power assay is a quantitative assay for measuring antioxidant potential present in the sample. Different concentrations of the extracts were prepared and results were measured with reference to standard the reducing power of the samples were comparable with the reference standard18.

 

DPPH free radical method:

1, 1-Diphenyl-2-picrylhydrazyl (DPPH) is a free radical, in presence of an antioxidant it gets reduced and becomes colorless or pale yellow color in the solution. DPPH is a well-known radical and a scavenger for other radicals hence it can be used to measure the reduction rate of the chemical reaction. Add 50µl of extract to 1.0 ml of 0.1ml of DPPH compound in NaOH solution. Incubate for 30 minutes in dark at 37 degrees centigrade. Measure absorbance at 517nm19.

 

Anti-microbial activity minimum inhibitory concentration (MIC) of C. Bipinnatus:

The antibacterial activity was carried out by employing 24h cultures with given compounds by using the Agar-Well diffusion method. The medium was sterilized by autoclaving at 120°C (15 psi) for about 30min. Standard (Ciprofloxacin) with 5μg/ml was used as a positive control. The activity diameter of the zone of inhibition was measured using the HiMedia antibiotic zone scale. The procedure is same for culturing of both Bacteria and Fungi. The difference is medium where Potato Dextrose is used for fungi and temperature for incubation is 28+2oC20

 

 

RESULTS AND DISCUSSION:

Discovering of drugs and screening of medicinal plants for their biological activities is an interesting and helpful to produce new drugs against various diseases for treatment. The dried material of different parts of C. bipinnatus have shown better yield when they were subjected to Soxhlet extraction followed by rota evaporation. The ethyl alcohol extracted leaf has shown yield of (6.7%) compared to the stem (5.3%) and flower (3.8%). The root was shown less yield with the ethyl alcohol. Similarly flower yield is high with ethyl acetate (5.3%) compared to leaf (5.2%) stem (4.7%) and root (1.1%). The hexane extracted leaf has better yield (4.9%) compared to stem (2.8%) and flower (4.2%) and root (1.3%). The results of preliminary phytochemical analysis by the three solvent extracts (ethanol, ethyl acetate and hexane) of different plant parts such as, root, stem, leaf and flower were presented in Table 1.

 

Table 1: Phytochemical analysis of the different parts of Cosmos bipinnatus in different solvents

Name of the Phyto chemical

Name of the test

 Leaf       

Stem

Flower

Root       

E

EA

H

E

EA

H

E

EA

H

E

EA

H

Phenol and alkoloid

Dragendroff’s test

-

-

-

-

+

+

+

+

+

-

-

-

 Mayer’s test

-

-

-

-

+

+

+

+

+

+

+

+

Steroid

Salkowski test

+=+

-

-

-

+

+

+

+

-

+

+

+

Liebermann buchards

+ =+

-

-

-

+

+

+

+

-

+

+

+

Terpenoids

Salkowski test

+

-

+

-

-

+

-

-

-

+

+

+

Liebermann buchards

+

-

+

-

-

+

-

-

-

+

+

+

Tannins

Ferric chloride test

+

-

+

-

-

+

-

-

-

+

+

-

Gelatin test

+

-

+

-

-

+

-

-

-

+

+

-

Flavonoids

Ferric chloride test

+

+

+

+

+

-

-

-

-

+

-

-

Lead acetate test

+

+

+

+

+

-

-

-

-

+

-

-

Saponines

Saponine test

+

-

-

+

-

+

-

+

+

-

-

-

Glycosides

Kellar-killianis test

-

-

+

+

+

-

-

+

-

+

+

+

Bornhaggers test

-

-

+

+

+

-

-

+

-

+

+

+

Carbohydrates

Molisch test

-

-

-

+

-

+

+

+

-

-

-

-

Fehlings test

-

-

-

+

-

+

+

+

-

-

-

-

Proteins

Biuret test

-

-

-

-

-

-

-

-

-

-

-

-

Aminoacids

Ninhydrin Test

-

-

-

-

-

-

-

-

-

-

-

-

Ethyl alcohol; EA: Ethyl acetate; H: Hexane; (-): absent; (+): present

 


The phytochemicals such as alkaloids, steroids, terpenoids, tannins, flavonoids, saponins, glycosides, carbohydrates, proteins and amino acids were observed in qualitative analysis. From the table, it is clear that carbohydrates, phenols and tannins, flavonoids and saponins were present in some parts of the plant. Flavonoids were abundantly found in the leaves and the root of C. bipinnatus. Similar results have been found in the research paper recently published by Sundaram Sowmya et al. It is stated that flavonoids were abundantly found in the leaf part of the plant Cayratia trifolia when they have conducted qualitative analysis on different parts of this plant21. Steroids were absent only in the leaves and flowers of C. bipinnatus while terpenoids were completely absent in the flowers of the plant. Alkaloids were absent in the leaves extracted from all the three solvents and root sample extracted using ethyl acetate and stem sample extracted using ethanol. Proteins were absent in all the parts of the plant extracted using all three solvents namely ethanol, ethyl acetate and hexane. Tannins were absent in the entirely in the flower in addition to the stem sample taken using the solvent hexane. Saponins were present in all the parts like leaf, stem and flower but are not found in the root extract of the plant. The detailed description of the various compounds present in the parts of the plant extracted using three different solvents. The results of total phenolic content of three different solvent extracted plant samples including the leaf, stem, root and flower were represented in the Figure 1.

 

For the plant samples extracted by using ethanol and hexane as the solvents, the concentration of phenol content was highest for the stem (740.32±0.52µg/mg GAE and 790.08±0.17µg/mgGAE) respectively. For the plant samples extracted by using ethyl acetate as the solvent, the concentration of phenols is highest for the root which was 661.04±1.03µg/mgGAE. Comparatively, in our study the phenolic content was found to be highest for the stem fraction that was extracted by using hexane as the solvent. Similar results have been obtained by Sengul, Memnune, et al., it was reported that the total phenolic content of Crocus sativus was found out to be 422.9µg GAE per mg dry weight basis22. In addition to the total phenolic content the results of total flavonoid content were presented in figure 2.

 

 

Figure 1: Total phenolic content of different parts of solvent extracted C. bipinnatus

 

 

Figure 2: Total flavonoid content of different parts of solvent extracted C. bipinnatus

 

For the plant samples extracted by using ethanol as the solvents, the total flavonoid content was found to be highest for the leaf 81µg/mg. Comparatively, the total flavonoid content of the leaves of Meyna spinosa plant was found out to be 58.5µg quercetin equivalents/mg23. For the plant samples extracted by using ethyl acetate as the solvent, the total flavonoid content was highest for the root which is 178.6µg/mg compared to the flower that is 125µg/mg. In this study the total flavonoid content was found to be highest for the root part extracted using ethyl acetate as the solvent. Polyphenolic compounds such as flavonoids and anthocyanins are of great interest for their radical scavenging activity. Intake of dietary antioxidants that act as radical scavengers is expected to be effective in preventing many diseases. The results of antioxidant activity by using the FRAP method was presented in figure 3.

 

Figure 3: Ferric acid reducing activity of different parts of solvent extracts of C. bipinnatus

 

The antioxidant activity of ethanol extracted root sample was highest when compared to ethanol and ethyl acetate fractions of root. The antioxidant activity of various parts of the plant Cosmos bipinnatus was tested using the DPPH assay at different concentrations (20, 60 and 120µg/ml). The percentages of inhibition for various extracts of the plant with different organic solvents were presented in figures 4,5 and 6.

 

 

Figure 4: Percentage of Inhibition of DPPH at 20µg/ml of different parts of solvent extracts of C. bipinnatus

 

 

Figure 5: Percentage of Inhibition of DPPH at 60µg/ml of different parts of solvent extracts of C. bipinnatus

 

Figure 6: Percentage of Inhibition of DPPH at 60µg/ml of different parts of solvent extracts of C. bipinnatus

 

At a concentration of 20µg/ml, the percentage of inhibition with respect to the solvent ethanol was lowest for the flower 52.87% compared to the percentage of inhibition with respect to the solvent ethyl acetate 59.09% and hexane was highest for the leaf 62.3%24,25,26. Taking all the solvents into consideration, the percentage of inhibition was highest for the leaf extracted using ethyl acetate. Similarly, at the concentration 60µg/ml, the percentage of inhibition with respect to the ethanol extract of flower was highest 91.42% compared to ethyl acetate of the root extract 92.16%. The percentage of inhibition with respect to the leaf in hexane extract was highest for compared to root 91.13% (Figure 4). The results of percentage of inhibition with respect to 120µg/ml of DPPH were shown in figure 5. The ethanol extracted root fraction has shown highest 91% compared to the percentage of inhibition with respect to the solvent ethyl acetate of root 87.7%. The percentage of inhibition with respect hexane fraction of root was highest 89% compared to ethyl acetate. Whereas the percentage of inhibition was highest for the flower extract of ethanol fraction compared to other solvent extracts. Similarly, in a study conducted by Ikhram ilahi et al. the percenat scavenging of stable DPPH free radical at a concentration of 60mg was found out to be 52.83% for the plant Withania somnifera27. The anti-microbial activity of all the extracts was determined by using both bacterial and fungal strains. The zone of inhibition was found and the results were presented in table 2. The fungal strains like Aspergillus niger and Trichophyton mentagrophytes were used28,29. The bacterial strains Staphylococcus aureus used as gram positive and dermatophyte Trichophyton mentagrophytes was also used. The zone of inhibition is calculated with the help of high media scale.

 

The antimicrobials from natural sources have drawn attention of many researchers to put an effort in order to identify novel compounds which can effectively act as suitable antimicrobials so as to replace the synthetic drugs. The phytochemicals derived from plant origin can serve as an effective prototype to develop less toxic and more effective drugs to control the growth of microorganisms30,31,32. Such compounds have significant therapeutic applications to combat the major human pathogens like bacteria, fungi and viruses. Several studies have been carried out with the extracts of various plant parts to identify the antimicrobial property as well as to discover new antimicrobials. The results of the current study states the therapeutic values of the C. Bipinnatus plant parts used in traditional medicine33. The antimicrobial activity reported in the current investigation is a scientific evidence for the traditional therapies and contains ample number of active compounds that can inhibit the growth of microorganisms. The presence of secondary metabolites in the extracts of different plant parts of C. Bipinnatus confirms the utilization of this medicinal herb in traditional medicine34,35.


 

Table 2: Antimicrobial activity of different parts of solvent extracts of C. Bipinnatus against various microorganisms

Name of the organism

Zone of inhibition mm (Mean± SD)

Solvent

Leaf

Stem

Flower

Root

Aspergillus

niger MTCC 961

Ethanol

3± 0.7

21± 0.8

10 ± 0.3

14± 0.5

Ethyl acetate

16± 0.1

20± 0.4

5± 0.6

4± 0.8

Hexane

11± 0.3

24± 0.6

19± 0.2

7± 0.3

Staphylococcus aureus

MTCC 3160

Ethanol

3± 0.2

3± 0.1

2± 0.2

5± 0.4

Ethyl acetate

8± 0.3

17± 0.2

8± 0.1

7± 0.2

Hexane

10± 0.4

22± 0.1

16± 0.2

6 ± 0.3

Klebsiella pneumonia MTCC 9024

Ethanol

10± 0.3

18± 0.2

15± 0.2

12± 0.1

Ethyl acetate

12± 0.4

14± 0.1

12± 0.5

7 ± 0.1

Hexane

8± 0.5

12± 0.4

8± 0.4

6± 0.2

Trichophyton mentagrophytes

MTCC 7687

Ethanol

14 ± 0.7

22± 0.1

9± 0.6

16± 0.3

Ethyl acetate

13± 0.1

18± 0.3

21± 0.4

26± 0.2

Hexane

10± 0.1

19± 0.1

17± 0.5

9± 0.1

 


CONCLUSION:

The phytochemical analysis of all the plant parts extracted using various organic solvents was done and the results were tabulated. The phytochemical study has shown the presence of several secondary metabolites like carbohydrates, phenols and tannins, flavonoids and saponins were present in some parts of the plant. Flavonoids were abundantly found in the leaves and the root of C. bipinnatus. The total phenolic and flavanoid content was observed in many parts of the plants. The antioxidant activity and antimicrobial activities of different parts of Cosmos bipinnatus in different solvents have shown good antioxidant and antimicrobial activities. The antimicrobial activity of extracts obtained from different parts of C. bipinnatus explains the reason behind the traditional use. Further separation of phyto chemical fractions for identification of biological active molecules can help to find solution against various diseases.

 

ACKNOWLEDGEMENT:

The authors also thankful to the Management of GITAM (Deemed to be University) for their constant support. The authors are thankful to Prof. Pandal, Dr. Prakash Rao Department of Botany Andhra University for identification of the plant.

 

CONFLICT OF INTREST:

Authors declared no conflict of interest

 

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Received on 24.09.2020            Modified on 19.06.2021

Accepted on 23.10.2021           © RJPT All right reserved

Research J. Pharm.and Tech 2022; 15(4):1455-1460.

DOI: 10.52711/0974-360X.2022.00241