Phytochemical Analysis and In vitro Anti-oxidant Activities of Medicinal Plants Cyperus rotundus, Tinospora cordifolia and their Formulation

Introduction

Oxidative stress results in the production of large amounts of highly reactive oxygen species, which cause DNA damage. The best-known free radicals that are highly toxic to cells are hydroxyl ions, hydrogen peroxide, superoxide, nitric oxide and oxides of nitrogen. There are many possible medicines available in nature. The World Health Organization states that drugs derived from plants are used in 80% of all traditional medical procedures worldwide. Indian traditional medical systems have mostly used the pharmacological analysis of several therapeutic plants¹. Natural bioactive substances known as “antioxidants” prevent oxidation by scavenging free radicals. Essential bioactive secondary metabolites produced by medicinal plants such as alkaloids, phenols and flavonoids can be used to treat a number of fatal degenerative diseases, notably cancer, cardiac issues and neurological disorders caused by oxidative stress ².

Generally two kinds of primary and secondary anti-oxidants are available, the primary antioxidant directly scavenging the free radicals and secondary antioxidant are indirectly impede the free radical production via fenton’s reaction. Plant extracts and natural products plays primary anti-oxidant role by scavenging free radicals. Antioxidants are applicable in many fields, such as food additives to protect food from deterioration, stabilizers for lubricants and fuels to delay or prevent oxidation, in gasolines to protect polymerization, which leads to engine-fouling residue formations and as drugs for cancer, asthma, diabetes, inflammatory joint diseases, atherosclerosis, degenerative eye disorders and senile dementia.

Cyperus rotundus, a member of the Cyperaceae family, can be found in tropical, subtropical, and temperate climates. It is commonly known as motha, purple nutsedge or nutgrass, Korai kizhangu in Tamil language ³. One of the useful medicinal plant parts used to cure nausea, diarrhoea, fever and inflammation is the rhizome of C. rotundus ⁴. It is a hazy medicinal herb that has been connected to an extensive range of effects, like analgesia, antibacterial activity, antidiarrheal activity, antidiabetic activity, anti-inflammatory activity⁵, antioxidant activity, appetiser, digestive and sedative effects ^6,7.

Tinospora cordifolia, also known as guduchi and “Shindilakodi,” in Tamil, is a member of the Menispermaceae family and is found all across India. It is a tall, climbing shrub with broad leaves. It has a number of health benefits, such as reducing inflammation, protecting the liver, boosting the immune system, lowering blood sugar and fighting cancer ^8,9.So, the aim of recent research is to find out whether C. rotundus and T. cordifolia extracts work as antioxidants.

Material and methods

Collection and Authentication of Plant Source

The rhizome part of C.rotundus and stem part of T.cordifolia were collected from the region of Lalgudi area, Tiruchirappalli district, Tamil Nadu, India. The obtained plant samples were verified as real, and voucher specimens were placed at the Rapinat Herbarium, located at St. Joseph’s College (autonomous), Tiruchirappalli, Tamil Nadu, India. The materials were collected, distilled water rinsed thrice and shade dried at room temperature. The pure plant materials were crushed into a coarse powder for further research.

Preparation of Plant Extract

The soxhlet hot extraction method was used to extract the phytocompound from the dried powder of C.rotundus and T.cordifolia using different organic solvents of water, ethanol, chloroform, ethyl acetate and petroleum ether. The different solvent extracts were made separately, for the plants C.rotundus and T.cordifolia as well as the combination of both plants (1:1 ratio).

Phytochemical Screening

 Different extracts of C. rotundus and T. cordifolia and its formulations were qualitatively screened for the phytoconstituents present ¹⁰.

Estimation of Total Phenolic And Flavonoid Content

Using Folin- ciocalteu method, the total phenolic content of rhizome of C. rotundus and stem of T. cordifolia, its formulations were measured ¹¹. Similarly, using the aluminium chloride method, the total flavonoid content of C.rotundus and T.cordifolia, as well as their formulations, was determined ¹².

In Vitro Antioxidant Assay

On the basis of a single antioxidant assay, the antioxidant activity of the plant extract should not be determined. In order to assess antioxidant activity, several in vitro test techniques such as the DPPH radical scavenging assay, ABTS radical scavenging activity, hydrogen peroxide scavenging activity, superoxide radical scavenging assay, and total anti-oxidant assay method are used. The extracts of C.rotundus and T.cordifolia and their formulations were tested against free radical scavenging properties. It determines the extract’s potential to neutralise the free radicals, and the change in optical density of radicals is observed in order to assess the antioxidant capability through free radical scavenging by the plant extract. ^{13,14,15,16,17}.The scavenging effect was calculated by using the formulae:

Scavenging effect (%) = (Absorbance of control – Absorbance of test solution or Standard) / Absorbance of control] × 100

Fourier Transform Infrared Spectrophotometer (Ft-Ir) Analysis

To prepare the sample disc for FT-IR spectroscopy, 10 mg of the sample was encapsulated in 100 mg of potassium bromide pellets ¹⁸. The extract’s functional groups absorbed light with a wavelength between 4000 and 400 cm^-1.

Results

Phytochemical Screening

A preliminary phytochemical analysis was done on the rhizome of C.rotundus, the stem extract of T.cordifolia and their mixtures. This result showed that phytochemicals were present in aqueous, ethanol, chloroform, ethyl acetate and petroleum ether extracts. These phytochemicals include flavonoids, diterpenes, proteins, saponins, amino acids and sugars (Table1).The phytochemical analysis of the C.rotundus rhizome revealed that the ethanolic and aqueous extracts had the highest amounts of various phytochemicals like alkaloids, flavonoids, steroids, tannins, phenols and saponins. Steroids, phenols and petroleum ether are also present in the chloroform, ethyl acetate and petroleum ether extracts of C.rotundus.

Phytochemical analysis of the stem of T.cordifolia revealed the presence of alkaloids, flavonoids, steroids, tannins, phenols and saponins in the aqueous and ethanolic extracts. Quinine, coumarins and glycosides were also present in the ethyl acetate and petroleum ether extracts of T. cordifolia. Alkaloids, flavonoids, steroids, tannins, quinine, coumarins, saponins and phenols were present in the formulations of C.rotundus and T.cordifolia. Tannin, quinine and phenols were more abundant in the aqueous and ethanolic extracts than the other three extracts studied. The ethanolic extract of C.rotundus and T.cordifolia were found to contain most of the phytochemicals.

Alkaloids, flavonoids, steroids, tannins, phenols and saponins were found in high amounts in the stem extracts of T.cordifolia (both aqueous and ethanol). Quinine, coumarins and glycosides were also found in the extracts of T. cordifolia in ethyl acetate and petroleum ether. The mixtures of C.rotundus and T.cordifolia had phenols, tannins, quinine, coumarins, flavonoids, alkaloids, and steroids. The aqueous and ethanolic extracts showed higher levels of tannin, quinine and phenols when compared to other three extracts. The aqueous and ethanolic extracts of C.rotundus and T. cordifolia were found to contain the majority of the phytochemicals.

Total phenolic content

The estimated phenolic content was expressed as gallic acid equivalent (GAE) per gram of plant extract. The highest concentration of total phenolic contents was observed 85.14± 7.82 mg GAE/g of dry extract in the formulation than the individual plant extract (58.27 ± 5.24, 45.43± 6.24 mg GAE/g of dry extract (Table 2).

Total flavonoids content

 Total flavonoid content was expressed as quercetin equivalent (QE) per gram of plant extract. The total flavonoid contents of C. rotundus and T. cordifolia extracts were found to be 110 ± 9.52 mg QE/g of dry extracts, respectively compared to the individual plant extracts (90.70 ± 8.96, 56.43 ± 6.94) in Table 2.

Table 1: Phytochemical Screening of C.rotundus and T.cordifolia and their formulations

S.No

Phytochemicals

C. rotundus

T.cordifolia

C. rotundus & T. cordifolia

A

E

C

EA

P

A

E

C

EA

P

A

E

C

EA

P

1

Alkaloids

–

+++

–

++

++

+++

+++

–

++

–

+++

+++

–

++

–

2

Flavonoids

+++

+

–

–

–

–

+++

–

++

–

+++

+++

–

–

–

3

Steroids

–

+++

+

++

++

+++

–

–

–

+

+++

+++

–

–

–

4

Tannins

+++

+++

–

–

++

+++

+++

–

–

–

+++

+++

++

–

++

5

Terpenoids

+++

+++

–

–

–

–

–

–

–

–

–

+++

–

–

–

6

Quinine

+++

–

–

++

–

+++

+++

–

++

++

+++

+++

++

++

++

7

Coumarins

+++

+++

–

–

–

+++

+++

–

++

–

+++

+++

–

–

–

8

Glycosides

+++

+++

–

–

–

+++

+++

++

++

++

–

–

–

–

–

9

Saponins

+++

+++

–

–

–

+++

–

–

–

–

+++

+++

–

–

++

10

Phenols

+++

+++

+

++

++

–

+++

++

++

–

+++

+++

++

++

++

Abbreviations: A-Aqueous, E-Ethanol, C-Chloroform, EA-Ethyl acetate, P-Petroleum Ether (+++)-High abundant, ++moderate abundant, + abundant,(-) negative

Table 2: Total Phenolic and Total flavonoid content of plant extracts

Extract	Total phenolic content (expressed as gallic acid equivalent (GAE) per gram of plant extract)	Total flavonoid content  (expressed as Quercetin equivalent (QE) per gram of plant extract)
C. rotundus	58.27 ± 5.24	90.70 ± 8.96
T. cordifolia	45.43± 6.24	56.43± 6.94
C.rotundus & T.cordifolia	85.14± 7.82	110± 9.52

 

DPPH free radical Scavenging Assay

This study also established the IC50 values for the different concentrations of plant extract required to scavenge 50% of the DPPH radicals. The IC50 values for the extracts of C.rotundus, T. cordifolia, C.rotundus and T.cordifolia combined were 250µg/ml, 300µg/ml, and 250µg/ml, respectively. Nevertheless, conventional ascorbic acid has an IC50 value of 250µg /mL (Table 3).

Table 3: DPPH Radical Scavenging activity of C.rotundus, T.cordifolia and its formulations

Con ( µg /ml)	% of Inhibition C.rotundus	% of Inhibition T. cordifolia	% of Inhibition C.rotundus & T.cordifolia	Standard Ascorbic Acid
50	12.90± 0.003	10.77±0.128	15.75±0.218	12.23±0.116
100	20.03±0.125	19.17±0.009	24.42±0.241	21.63±0.104
150	31.37±0.007	28.78±0.165	36.26±0.330	34.41±0.234
200	40.36±0.125	37.99±0.214	46.30±0.561	44.49±0.486
250	50.18±0.413	47.75±0.416	56.75±0.431	54.31±0.452
300	59.95±0.312	58.74±0.428	65.56±0.543	60.29±0.375
350	67.08±0.413	64.79±0.585	73.86±0.654	67.00±0.621
IC50 value	250.285	263.753	233.283	227.490

 

ABTS radical scavenging activity

At 300 g/ml, the highest percentage of inhibitory activity for C.rotundus was 58.68% and the highest percentage for T.cordifolia was 55.97%.The combination of plant extracts has the highest activity (54.9%) at 250µg /ml concentration and the activity of conventional ascorbic acid is 57.14% at 300µg /ml. These outcomes showed the potential activity of a plant extract formulation (Table 4).

Table 4: ABTS radical scavenging activity of C.rotundus, T.cordifolia and its formulations

Concentration of the Sample (µg /ml)		% of Inhibition C. rotundus	% of Inhibition T. cordifolia	% of Inhibition C.rotundus & T.cordifolia	Ascorbic acid
50		13.62 ± 0.021	13.31± 0.020	17.5 ± 0.018	13.88 ± 0.021
	100	16.47 ± 0.052	17.78 ± 0.047	24.2 ± 0.021	15.85 ± 0.031
150		23.77 ± 0.327	25.90 ± 0.031	35.2 ± 0.036	27.85 ± 0.034
200		38.09 ± 0.361	34.66 ± 0.030	42.9 ± 0.031	35.44 ± 0.029
250		44.84 ± 0.024	44.32 ± 0.029	55.9  ± 0.025	45.06 ± 0.027
300		58.68 ± 0.037	55.97 ± 0.032	64.6 ± 0.038	57.14 ± 0.037
350		68.65 ± 0.035	60.71 ± 0.026	76.0 ± 0.027	63.97 ± 0.031
IC50 value		284.67	294.52	227.80	286.57

Hydrogen Peroxide Scavenging Activity (H₂O₂)

The activity of plant extracts on hydrogen peroxide scavenging assay were found to be  maximum in  the combination of plant extract (248.91±25μg/ml).The results revealed that the ethanolic extracts of C.rotundus and T.cordifolia have moderate scavenging activity (61.60 ± 5.78%, 58.86 ± 4.32%) at a high concentration of 350µg/ml. The combination of herbal extracts (85%) had more potent hydrogen peroxide scavenging activity than the standard reference of ascorbic acid (Table 5).

Table 5: Hydrogen Peroxide scavenging activity of C.rotundus, T.cordifolia and its formulations

Con  ( µg /ml)	% of Inhibition C.rotundus	% of Inhibition T. cordifolia	% of Inhibition C.rotundus &T.cordifolia	Ascorbic acid
50	13.57 ± 0.021	13.27 ± 0.138	13.72 ± 0.321	13.29± 0.021
100	14.61 ± 0.352	14.48 ± 0.216	21.42 ± 0.392	14.58± 0.234
150	23.88 ± 0.427	21.50 ±0.354	29.71 ± 0.472	24.92± 0.354
200	32.95 ± 0.261	31.38 ± 0.482	39.91 ± 0.257	31.50± 0.432
250	43.81 ± 0.372	42.8 ± 0.672	51.74 ± 0.484	49.03 ±0.237
300	51.69 ±0.432	48.9 ±0.457	60.18 ± 0.419	48.86 ±0.413
350	61.60 ±0.278	58.86 ±0.432	68.64 ± 0.356	65.60 ±0.285
IC50 value	303.743	316.627	248.91	281.991

 

Superoxide radical scavenging activity

The results suggested that the ethanolic extract of C. rotundus, T. cordifolia and their combinations were greater scavengers of superoxide with a value of 55.28 ± 4.3%, 55.17± 4.72 %, and 57.76 ± 5.324 % at a concentration of 250µg/ml compared to the ethanolic extract of C. rotundus, T. cordifolia at the same concentration (Table 6).

Table 6 : Superoxide radical scavenging activity of C.rotundus, T.cordifolia and its formulations

Concentration  of the Sample ( µg /ml)		% of Inhibition C.rotundus	% of Inhibition T. cordifolia	% of Inhibition C.rotundus & T.cordifolia	Ascorbic acid
50		19.46 ± 0.021	20.22± 0.038	23.97 ±0.026	21.86± 0.023
	100	27.83± 0.052	30.01± 0.016	31.75 ± 0.192	26.87± 0.354
150		39.54± 0.327	40.43±0.254	42.42 ± 0.172	41.29±0.218
200		46.40± 0.361	46.35± 0.382	48.59 ± 0.257	47.73± 0.432
250		55.28±0.243	55.17±0.472	57.76 ± 0.324	56.61±0.340
300		64.82±0.0375	62.11±0.371	65.29±0.276	64.2 ±0.410
350		68.65±0.256	68.96±0.287	70.68 ± 0.327	69.21 ±0.347
IC50 value		218.163	217.045	203.909	211.576

 

Total antioxidant activity

Total antioxidant activity of ethanolic extract of C. rotundus, T. cordifolia and its formulations was carried out by phosphomolybdenum method (Table 7). The total antioxidant activity is expressed as equivalent to ascorbic acid. The results indicated the dose dependent increase in antioxidant activity at concentration 50 to 350 μg/ml. The strong antioxidant activity of combinations of these plant extracts was higher 93.34 % at 350μg/ml. The individual plant extracts of C. rotundus, T. cordifolia exhibited 89.91% at 350μg/ml, 90.45% at 350μg/ml respectively. The presence of high flavonoid and phenolic contents might be the reason for the antioxidant capacity of medicinal plants.

Table 7: Total antioxidant activity of C.rotundus and T.cordifolia and its formulations

Con ( µg /ml)	% of C. rotundus	% T. cordifolia	% of C. rotundus & T.cordifolia	% of Ascorbic acid
50	30.18	24.18	40.18	30.35
100	40.17	40.13	50.02	42.23
150	50.02	51.14	65.17	52.18
200	61.18	64.19	75.19	65.30
250	69.98	70.64	80.14	70.16
300	70.04	82.35	90.05	80.06
350	89.91	90.45	93.34	85.15

 

FT-IR ANALYSIS OF C. rotundus

FT-IR analysis was done on the ethanolic extract of C. rotundus to determine the potential biomolecules. C. rotundus has a lot of absorption bands, which means it has a lot of functional groups. While some intensity peaks, like those for the periods 1638 cm^-1 and 684 cm^-1 are decreased, others like those for the periods 3363 cm^-1, 2114 cm^-1, 1641 cm^-1 and 1277 cm^-1 are greatly enhanced. The band at 3363 cm^-1, as shown in Fig. 1, depicts N-H¹, the stretching vibrations of primary, secondary and tertiary amines and amides. At 2114 cm^-1, the C-N Stretch in plane bend to Nitriles was depicted. The peak at 1641 cm^-1 is caused by the C=O stretching vibrations to carbonyls. The presence of alkyl halides in the C. rotundus extract is consistent with the faint band at 684 cm^-1, which denotes CBr Stretch vibrations.

FTIR ANALYSIS OF T. cordifolia

The graph showed the ethanolic extract of T. cordifolia peak value and functional groups from the FTIR spectrum. Strong intensity peaks at 3362 cm^-1  and 2128 cm^-1 were visible in this spectra, as well as smaller peaks at 1640 cm^-1 and 558 cm^-1, which respectively denote the presence of primary amines as well as secondary amines, nitriles, alkenes, nitro compounds and alkyl halides (Fig. 2).

FTIR ANALYSIS OF C. rotundus and T.cordifolia 

Figure 3 displays the FT-IR spectrum’s results. The large, noticeable peak at 3369 cm^-1 and 2126 cm^-1, which correlates to the presence of alkene compounds, is illustrated in this spectrum of formulations of C. rotundus and T. cordifolia (fig.3). The signal at 1642 cm^-1 similarly denotes the presence of nitro compounds. The faint band at 668 cm^-1 indicates that alkynes are present in the formulation.

Figure 1: FT-IR ANALYSIS OF C. rotundus Click here to View Figure

Figure 2: FT-IR ANALYSIS OF T. cordifolia Click here to View Figure

Figure 3: FT-IR ANALYSIS OF C. rotundus and T. cordifolia formulation Click here to View Figure

Discussions

Plants contain naturally occurring substances known as “phytochemicals.” Due to their antioxidant or disease-preventing actions, the secondary metabolites, such as alkaloids, tannins, flavonoids, steroids, terpenoids, carbohydrates and phenolic compounds, have numerous therapeutic capabilities, such as in the instances of heart disease, cancer, hypertension and diabetes ¹⁹. Alkaloids act as analgesic and antispasmodic agents, while saponins have antimicrobial and antifungal properties. The bioactive compounds namely alkaloids, saponins, flavonoids and phenolic compounds acts as a defense mechanism in humans to inhibit degenerative diseases and has pharmacological activity, which includes free radical scavenging activity, anti-inflammatory activity and cytotoxicity ²⁰.

Phenolic compounds and flavonoids like flavones and flavanols have redox potentials that enable them to provide free radicals with hydrogen atoms; they act as antioxidants. These substances are crucial for the prevention of illnesses connected to oxidative stress ²¹. The observations of this investigation demonstrated that the high levels of polyphenols and flavonoids in the ethanolic extracts of C. rotundus and T. cordifolia gave rise to the highest antioxidant capacity ²².

Reactive oxygen species are produced as a result of biological metabolism, the most prevalent reactive oxygen species are superoxide radicals, singlet oxygen, hydrogen peroxide and hydroxyl radicals ²³. Reactive species may be causing DNA mutations that result in tissue damage, cancer and neurogenic illness through oxidation. Moreover, it performs a number of significant activities, including suppressing tumors, delaying cell damage and enhancing cellular defenses against different diseases. Antioxidants protect cells from the ailment inducing effects of free radicals ²⁴. The DPPH, superoxide radical scavenging assay, hydrogen peroxide scavenging activity and total antioxidant activity were used in the current investigation to measure the in vitro antioxidant activity of plant extract.

The most commonly used method for determining a plant’s capacity for antioxidants is the DPPH radical scavenging assay. In vitro DPPH radical activity of the ethanolic extracts of C. rotundus and T.cordifolia was evaluated. The present results suggest that plant extracts’ phytochemicals have the ability to scavenge free radicals and lower the risk of oxidative diseases. The previous research explained the antioxidant activity of plant extract by measuring the ability to bring down free radicals and expressed in terms of hydrogen atom donating ability ²⁵.

An important and widely used technique to assess the antioxidant capacity of plant extracts is the ABTS free radical scavenging assay. The antioxidant effect of a plant extract needs ABTS to lose its color, which also makes free radical species ineffective. At 734 nm, the average absorbance was measured, which made it harder for proton radicals to get rid of extra electrons. These findings demonstrated that formulations of C. rotundus and T. cordifolia had effective free radical scavenging effects against ABTS free radicals. The previous study reported the dose dependent increase in the in vitro scavenging activities of methanolic extracts of Caesalpini avolkensii Harms. Vernonia lasiopus O. Hoffm.,  and Acacia hockii De Wild ²⁶.

A weak oxidizing agent, hydrogen peroxide, instantly inactivates the enzyme. A reactive free radical formed in the biological system can easily cross the cell membrane. The generation of hydroxyl radicals interacts with transition metal ions like Fe ²⁺ and Cu ²⁺ to severely damage cells ²⁷. The interaction of antioxidant chemicals with hydrogen peroxide neutralizes it. In comparison to the ascorbic acid standard reference, the mixture of C. rotundus and T. cordifolia (85%) demonstrated strong hydrogen peroxide scavenging action. Similarly previous study reported the higher potency free radical scavenging activity of different leaf extracts from Kedrostis foetidissima (Jacq.) ²⁸.

Superoxide anion is harmful free radical to the cellular components produced by the incomplete metabolism of oxygen. The inefficient use of oxygen in the body makes superoxide anion, which is a dangerous free radical for biological parts. As a result, it also causes harm to biomolecules by causing the production of H₂0₂, OH, peroxynitrite, or singlet oxygen. To keep superoxide radicals from hurting the cells, they must be neutralized ²⁹. Due to the presence of flavonoids, the study’s findings showed that the mixture of plant extracts had excellent potential for creating superoxide radicals. Recently, research reported that the crude extract of solanum nigrum linn shows high degree of super oxide radical scavenging activity ³⁰.

Poly phenolic like flavonoids, phenolic acids and tannins are regarded as powerful antioxidant of plant extract. These compounds are considered as reducing agents and free radical quenchers that exhibited various medicinal properties like antibacterial, antiviral and antidiabetic activities ³¹. In our study, the ethanolic extract of formulation of plant extract exhibited high antioxidant potential compared to the reference standard. Scavenging potential of the plant extract also depends on the high content of natural polyphenol compounds present in the plant extract ³².

The most important method for identifying the functional groups of the bioactive components found in the plant extract is FT-IR spectroscopic analysis. The ethanolic extracts of C. rotundus and T. cordifolia contained alkenes, alcohols, alkanes, alkyl halides and aldehydes, according to the findings of FT-IR analysis ³³.

Conclusion

From the overall observations, it is to state that the ethanolic extracts of C. rotundus and T. cordifolia exhibited the maximum free radical scavenging activities in a synergistic way in an in vitro model. Hence, further researches are required to explore the therapeutic value of Cyperus rotundus and Tinospora cordifolia.

Conflict of Interest

There is no conflict of interest.

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