Structure Analysis and Antimicrobial property of Tinospora cordifolia leaves extract

 

Tharani S, Mathusree I, Pradeepa S Y, Sreeraj S, Divya Nair, Bindhu J*

Department of Biotechnology, Sri Shakthi Institute of Engineering and Technology, Coimbatore.

*Corresponding Author E-mail: bindhuravi.edu@gmail.com

 

ABSTRACT:

Introduction: A review of pharma and chemical constituents found in the various part of Tinospora is given in the present articles. Tinospora cortisol is also commonly known as Guduchi is known for the application of treatment of many diseases in the ayurvedic literature. T.cordifolia is the best for those children who are all suffering from respiratory tract infection. The review of the present article shows all the updated information on its phytochemical and pharmacological activities which are performed by different methods. It also shows possesses antioxidant, antibacterial and also shows hepatoprotective properties. Objective: The main objective was to study the qualitative preliminary analysis of Tinospora cordifolia. It is a well-known medicinal plant with lots of medicinal properties. Identifying the active compounds and their role from this plant leads its importance. Methods: It shows all the updated information on its phytochemical and pharmacological activities which are performed by different methods by extracting leaves of T.cordifoliato perform phytochemical tests, Gas chromatography-mass spectroscopy (GC-MS), Fourier transform infrared spectroscopy (FTIR) analysis and antimicrobial test. And the results showed variety of active components derived from the plant like alkaloids, steroids, aliphatics, and glycoside etc. Where Antimicrobial property was also performed with gram-positive and gram-negative bacteria. Results: Laboratory studies shows that plant used for treatment of cold, diabetes, rheumatoid arthritis, fever and showed significant anticancer activity. Conclusion: T.cordifoliain drug industry as a cure to many life threatening diseases, Scientist are using phytodiversity to produce nanoparticles which plays an important role in drug delivery, diagnostic imaging, sensing, gene delivery.

 

KEYWORDS: T.inosporacordifoli, GC-MS, FTIR, Leaves extract.

 

 


INTRODUCTION: 

Medicinal plants are playing one of the most important roles in the human field in terms of healthcare all over the world. In ancient times herbalists used many other herbs and minerals such as Charaka and Sushruta during the first millennium BC.1 Ayurveda alone mentions the use of close to 1000 plants with Susruta Samhita mentioning around 770 plants used in the 1st millennium BC in India as medicine. There are estimates of 500,000 plants having medicinal properties of which 3000 plants have the potential use in cancer treatment. Among all these medicinal plants, Tinospora cordifolia (wild) Hook. F and Thomson of family Menispereaceae is a very valuable plant in terms of pharmacology.

 

The family Menispereaceae contain a total of 70 Genus and 450 species. This family is rich in alkaloids, terpenes, steroids, glycosides, and aliphatic.2

 

Medicinal Property:

The plant possesses anti-oxidant, anti-neoplastic, anti-stress, ant-allergic, antipyretic, anti-hyperlipidaemia properties. Guduchi is an Ayurveda medicine, and used in the treatment of fever, jaundice, diabetes, chronic3-4 diarrhoea, urinary disorders and cancer.6 Guduchi is useful for treating the patients having heart dieases, rheumatoid arthritis and leprosy. The tribal living in Baiga area of Naugarh and Uttar Pradesh they are making paste of the stem and preparing pills for the treatment of fever. For the treatment of the pain in ear two drops of leaf juice can reduce the pain. By making the powder of T.cordifolia and Terminalia chebula in equal quantity, orally daily once in morning with salt can be used for the treatment of Kasa (cough)6. In recent years, because of the concerns on the safety against synthetic drugs the use of natural antioxidants has been very promoted. Antioxidant plays an important role in human’s life by protecting the cell damage by reactive oxygen species and reducing the adverse effects of these free radicals7. These free radicals will behave like a primary catalyst for oxidation in, in vitro and vivo therefore oxidative stress will be there which will cause many diseases like Parkinsons disease, ageing etc.8

 

MATERIALS AND METHODS:

Identification and Handling of Sample:

Plants collection:

The stem of Tinospora cordifolia  is basically found in the local market of Karantaka i.e. Udupi market and some part of Tamil nadu i.e. in a small city called Sathyamangalam.

 

Extraction of selected plant material powder by:

The fresh plant of T.cordifolia was collected and was air-dried under 37°C for 72hours and it was grinded to a fine powder and in an air-tight container it was stored, exposure of sunlight was completely avoid.

 

Reagents and chemical:

Extraction of extract and distillation:

The dried powered stem and leaf of T.cordifolia were exhaustively extracted with absolute ethanol by distillation method at temperature 60°C and keep it in bathwater for 1hour at 50°C for further use i.e. for GC-MS.9

 

Preparation of plant extract:

For the extraction of phytochemicals, 8gm of powder was taken on 4 centrifuge tubes and it was filled with 2gm of powder and then it was filled with distilled water and centrifuge it at 10,000rpm for 20min to get the extract for the further test.

 

Preliminary qualitative phytochemical screening:

To detect the presence of secondary metabolites viz alkaloids, lipids, steroids, terpenoids, triterpenoids, phlobaianninls, glucosides, saponin, flavonoids, Proteins, Phenol. The presence or absence of the phytochemical constituents was analyzed by using these following protocols:

 

Test for alkaloids:

Mayer's test – 3ml of extract and 1ml of Mayer reagents, white colour Represent the indication

 

Test for lipids:

Take 0.5ml extract 0.5N alcoholic KOH add with phenolphthalein, heat it for 1 hours, indication will be soap formation

 

 

Test for steroids:

Take 2ml of chloroform and conc H2SO4 add in the 5ml

of plant extract, the test tube turns red that is the presence of steroids

 

Test for Terpenoids:

Take 1ml extract add 1ml of chloroform and add 1ml conc H2SO4 after 5min, grayish colour indicates the presence.

 

Test of phlobaianninls:

Take 10ml of aq extract of plants is boiled with 1% HCL, Red precipitate colour indicates the presence.

 

Test for glucosides:

Take 1ml extract, 0.5ml glacial acid, 2 drop of 5% chloride and conc sulphuric acid, red brown colour indicates presence

 

Test for Saponins:

Take 20ml H20 and 1ml extract, foam indicates the presence

 

Test for flavonoids:

Take dry extract 2g, 5ml of ethanol drops and HCL and 0.5g of magnesium, pink colour indicates the presence.

 

Test for proteins:

Take 3ml extract, 3 drops of 5% dead acetate solutions, boiled for 10min, purple colour will indicate the presence

 

Test of phenol:

1ml extract, 1ml of 5% ferric chloride add, Blue black indicates the presence.

 

GC-MS analysis:

The Clarus 680 GC was used in the analysis employed a fused silica column, packed with Elite-5MS (5% biphenyl 95% dimethylpolysiloxane, 30m × 0.25mm ID × 250μm df) and the components were separated using Helium as carrier gas at a constant flow of 1ml/min. The injector temperature was set at 260°C during the chromatographic run. The 1μL of extract sample injected into the instrument the oven temperature was as follows: 60°C (2 min); followed by 300°C at the rate of 10°C min1; and 300°C, where it was held for 6 min. The mass detector conditions were: transfer line temperature 230°C; ion source temperature 230°C; and ionization mode electron impact at 70 eV, scan time 0.2 sec, and scan interval of 0.1sec. The fragments from 40 to 600Da.10 The spectrums of the components were compared with the database of the spectrum of known components stored in the GC-MS NIST (2008) library.

 

Antibacterial assay:

The indicator organisms were procured from Microbial Type Culture Collection, Chandigarh and National Collection of Industrial Microorganisms, Pune, for antibacterial testing. The antibacterial assay of Carica papaya was performed using five gram-positive bacteria like Listeria monocytogenes, Bacillus subtilis, Streptococcus agalactiae, Staphylococcus aureus, Streptococcus faecalis and 3 gram-negative bacteria like Escherichia coli, Klebsiella oxytoca, Klebsiella aerogenes by following well diffusion agar method11. The following bacteria was inoculated in sterile nutrient plates individually in triplicates by using swabbing technique. The wells were made in the diameter of 3.5 mm using the sterile agar well puncher. The leaf extract sample was diluted at different concentration (20%, 40%, 60%, 80% and 100%) using distilled water. Then the agar plates were incubated at ±37˚C. After 24hours of incubation, the zone of inhibition was measured in mm.11

 

Antioxidant activity:

The antioxidant activity of the extract was determined by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay. Both the samples and standards were taken in different concentrations and the volume was adjusted to about 100 l using methanol. The DPPH solution of 0.1mM was prepared by using methanol as a solvent12. About 3ml of 0.1mM DPPH solution was mixed with samples of different concentrations and the negative control was prepared by adding 100 l of methanol to the 3ml of the solution.13 The tubes along with mixtures were allowed to stand in dark at room temperature for 30mins. The change in shading from violet to yellow shows the nearness of cancer prevention agents and the evaluation was by estimating its absorbance at 517nm against the clear14. The IC50 esteem (inhibitory focus) was determined for both sample and standard. The level of hindrance was determined to utilize the accompanying recipe.15

 

% of hindrance = [A0 - A1/A0] *100

Where A0 is the absorbance of the control (for example DPPH arrangement without test) and A1 is the absorbance of a test or on the other hand standard (for example DPPH arrangement with test/standard.

 

RESULTS AND DISCUSSION:

Research have shown that fruit, bark, stem, and leaves of cannonball tree are effective for the treatment of High Blood Pressure, Malaria, Stomach Ache, Tumours, Inflammation, Pain, Toothache, Wounds, etc., They are used as Analgesic, Antifungal, Antimalarial, Antiseptic, Local Antibiotic, Anti Inflammatory, and Anti-cancerous agents.16 The effective nutrients that are present in the cannonball tree are Couroupitone, Henolic Substances, Isatin, Volatile Oil, Glycosides, Indirubcin, and Keto Steroids17. Chewing of cannonball leaves helps in the curing of toothache. The flesh of cannonball fruit acts as a local curative for wounds18. The leaf extract treats skin diseases. The unripe fruit is harmful and might cause an allergic reaction. The isatin present in the flower of Couroupitaguianensis is known to have cytotoxicity against carcinoma cell lines19. This compound has potential as a chemotherapeutic agent. They show anticancer activity and cytotoxicity against HL60 cells. The leaf extract was screened against six human pathogenic bacteria and four fungal strains.15 This shows the antimicrobial activity if the plant. The antiulcer activity of the plant is reported by Elumalai et al says that the ethanolic extract of Couroupita Guienensis inhibits the formation of the gastric lesion. Anti-inflammatory activity is reported by Pinheiro et al as the ethanolic extract was able to inhibit the leucocyte migration into the peripheral cavity20. Stigmasterol and flavonoids present in the extract have antioxidant which decreases the time of wound healing process. The phytochemical studies showed the presence of proteins, tannins and phenols, glycosides, Alkaloids, and flavonoids whereas it shows the absence of saponins, Triterpenoids, steroids, terpenoids in the aqueous extract. Triterpenoids and terpenoids were absent in the ethanol extract whereas all others are present. The results were shown in Table 1.

 

Table 1: The phytochemical constituents of Tinospora cordifolia

Phytochemical constituents

Presence

Lipids

-

Flavonoids

-

Alkaloids

+

Glycosides

 +

Proteins

 -

Terpernoids

 +

Phenol

 -

Steroid

 +

Saponins

 -

Phlobaianninls

 -

 

The glycosides have Anti- inflammatory, anti-cancer and immune boosting properties. The alkaloids have antimicrobial properties whereas the flavonoids have anti-oxidant properties.

 

The result of GC-MS is shown in the Table 2 and the components found from the leaf extract. In this table, the components that are found during GC-MS analysis are shown in Figure 1.


 

 

Table 2: GCMS analysis result for major phytocomponents in Tinospora cordifolia extract

Sl. No

RT

Name of the compound

Molecular formula

Molecular weight

Peak area

%

Structure

 

1.

18.665

 

N-Hexadecanoic

ACID

C16H32 O2

256

2.872

 

2.

19.935

9,12-

Octadecadienoic Acid

C18H32O2

280

8.324

 

3.

20.641

Hexatriacontane

C36H74

506

10.633

 

4.

21.366

Tritetracontane

C43H88

604

20.037

 

5.

22.186

Heptacosane

C27H56

380

21.835

 

 


 

Fig. 1:- Chromatogram of compounds in the Tinospora cordifolia

 

The Chromatogram graph that is given below is shown that molecules that have the highest peaks are Hexatriacontane, Octacosane. The molecule N-Hexadecanoic acid, 9,12-Octadecadienoic acid is the oxygen-carrying molecules which means they behave antioxidant and the other molecules have anti-microbial, anti-cancer, anti-bacterial properties.FTIR spectrum indicating functional groups are indicated in Figure 2.

 

 

Fig 2: FTIR- analysis result for the phytochemical compounds found in the Tinospora cordifoli

 

The antimicrobial properties were carried out to identify whether the extract undergoes antibacterial activity20. The well diffusion assay was also performed to find the antibacterial properties. The clear region around the well exhibits no bacterial growth and this is called the zone of inhibition21. The different variety of bacterial strains which were used to perform the antibacterial activity are Listeria monocytogenes, Bacillus subtilis, Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli, Streptococcus faecalis, Klebsiella aerogenes, Klebsiella oxytoca. Among all these strains, Escherichia coli and Streptococcus faecalis have shown the highest susceptibility to the plant extract. (Figure 3), Klebsiella has exhibited moderate sensitivity. The bacterial species  Listeria monocytogenes, Streptococcus agalactiae showed lower susceptibility21.

 

Listeria monocytogenes showing least zone of inhibition

 

 

E. coli showing the highest zone of inhibition

Figure 3: Antibacterial activity by well diffusion method, Concentration of extract vs Zones of inhibition against bacteria in mm

 

DPPH (2, 2-diphenyl-1-picryl-hydrazyl-hydrate) is a process to detect the antioxidant activity (fig: 3). generally, all the plants have antioxidant activity which is responsible for the arrest of various reactive species as a result of DNA damage21. Ascorbic acid was used as the reference for comparison with the samples of different concentrations and IC50 (inhibitory concentration) was measured for both sample and the standard graph shows the anti-oxidation characteristics versus Tinospora cordifolia (sample) concentration.

 

DECLARATION OF INTEREST:

The authors report no conflicts of interest

 

ETHICAL CLEARANCE:

Ethical approval is taken from IEC. This work is carried out by following the strict guidelines of IEC.

 

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Received on 23.07.2022            Modified on 27.11.2022

Accepted on 25.03.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(10):4836-4840.

DOI: 10.52711/0974-360X.2023.00784