INTRODUCTION:

Cissampelos pareira is one of the few plant species that has garnered the interest and reverence of herbalists, scientists, and traditional healers in the same measure as it has in the field of plant-based medicines. This unassuming vine, with its inconspicuous presence in the biodiversity-rich landscapes of tropical and subtropical regions, holds within its botanical lineage a treasure trove of healing potential¹.

Some of the common names for this plant include "velvetleaf," "abuta," and "midnight horror." Since ancient times, Cissampelos pareira has played a pivotal role in the development of traditional medical practises all across the world. This plant has left an unmistakable stamp in the annals of herbal learning, from the Ayurvedic practitioners of the Indian subcontinent to the indigenous healers of the Amazon rainforests. Its significance, which is firmly ingrained in cultural traditions and has been passed down through generations, serves as a witness to the flexibility of its therapeutic applications².

Not only does the historical significance of Cissampelos pareira add to its appeal, but so does the scientific mystery that surrounds it. In recent decades, the fields of phytochemistry and pharmacology have opened up new areas of inquiry, shedding light on the complex biochemical makeup of this botanical marvel and the medicinal promise it possesses. Researchers are focusing a lot of attention on the phytochemical makeup of this plant, which includes a wide variety of alkaloids, flavonoids, terpenes, and phenolic compounds. They are doing this because they want to learn more about the plant's medical capabilities. Equally fascinating are the pharmacological aspects of Cissampelos pareira. Historically, this plant has been used to cure a wide variety of health conditions, ranging from gastrointestinal illnesses and respiratory diseases to more complex health issues; these applications were frequently guided by the experiential knowledge of indigenous societies^3,4. In the framework of contemporary pharmacology, we now have access to the resources and approaches necessary to solve the riddles surrounding its various therapeutic processes. Investigations conducted in the realm of science have uncovered, among many other fascinating qualities of the plant, its ability to act as an anti-inflammatory, analgesic, antibacterial, antioxidant, and immune-modulatory agent⁵. As we set off on this fascinating voyage through the phytochemistry and pharmacology of Cissampelos pareira, we enter a world in which time-honored knowledge and cutting-edge research coexist⁶. Cissampelos pareira has a wide variety of bioactive components like hayatine, cycloline, cycleanine, quercetin and kaempferol that have significant anti-inflammatory and wound-healing properties . Moreover, Cissampelos pareira to modify a variety of various pathways is the source of the plant's anti-inflammatory properties, which also contribute to the plant's reputation. Both cyclooxygenase-2 (COX-2) and lipoxygenase (LOX) are enzymes that can be inhibited by this substance with the capacity to reduce their activity. In the process of producing pro-inflammatory mediators like prostaglandins and leukotrienes, these enzymes play a role in the production of these substances. The anti-inflammatory effects of Cissampelos pareira have demonstrated some potential for treating inflammatory diseases such as rheumatoid arthritis. In the treatment of arthritis, a lot of attention has been focused on its ability to reduce joint swelling as well as pain⁷. In addition to this, it is able to block the nuclear factor-kappa B (NF-B) pathway, which is an essential component in the regulation of inflammation⁸ This plant provide a natural and complementary approach to traditional pharmaceuticals, offering alternative possibilities for persons who are looking for effective and holistic solutions for the control of various disorders.

METHODOLOGY:

Chemicals and plant material

The substances that were utilised included carrageenan, diagnostic kits, and analytical grade for all others. These chemicals were bought from HiMedia: methanol, chloroform, ethanol, sodium carbonate, Folin-Ciocalteau reagent, aluminium chloride, MTT (3-(4,5-dimethyl-2- thiazolyl)-2,5diphenyl-2H-tetrazoliumbromide), and cell culture grade dimethyl sulfoxide (DMSO) (Mumbai, India). We bought MSTFA and DPPH (1,1-diphenyl-2-picrylhydrazyl) from Sigma (Bangalore, India). The Cissampelos pareira was authenticated from the plant authentication cell of Guru Ghasidas Vishwavidhyalaya, Bilaspur, Chattishgarh-495009, India with authentication number Bot/GGV/2022/20.

Preparation of extracts

In order to ensure that each component of the plant was thoroughly cleaned, dried, and pulverized, a mixer grinder was utilized. The preparation of numerous extracts, ranging from nonpolar to polar, were accomplished by the use of sequential solvents. These extracts included methanol and aqueous extract. After being concentrated with a rotary evaporator, the extracts were kept in airtight containers at a temperature of 4 degrees Celsius for use in subsequent experimental study^9,10.

Preliminary phytochemical analysis

The preliminary qualitative phytochemical examination was carried out with the purpose of identifying several phytoconstituents, including alkaloids, phenols, flavonoids, tannins, and terpenoids, by employing the relevant tests for each phytochemical^11,12 .

In-vitro antioxidant activity

I. Assay of reducing power

In order to determine the reducing power, a mixture was prepared. This combination consisted of 2.5mL of sample with varying concentrations ranging from 20 to 320μg/mL, 2.5mL of 0.1M sodium phosphate buffer with a pH of 6.6, and 2.5mL of K3Fe(CN) 6(1%, weight-to-volume). The mixture was then incubated at a temperature of 50°C for a duration of 20 minutes. After adding 2.5ml of trichloroacetic acid with a weight-to-volume ratio of 10%, the mixture was centrifuged at 5000rpm for ten minutes. In the following step, the absorbance was measured at 700nm after the upper layer, which was 4ml, was combined with 0.4ml of new FeCl3 that was 0.1% by weight. The value of the inhibitory concentration (IC50) was determined by using ascorbic acid as the standard¹³.

II. Assay of H₂O₂ scavenging activity

As part of this particular experiment, the extract of varying concentrations, ranging from 20 to 320μg/mL, was combined with 2.5ml of 0.1M phosphate buffer (pH 7.4) and 600μl of 41mM of H₂O₂ solution. The mixture was then vigorously shaken and incubated at room temperature for a duration of ten minutes. Afterwards, the absorbance of the reaction mixture was measured at 230nm to ascertain its value. At the same time, VC served as the positive control. A calculation was made to determine the H₂O₂ scavenging activity as follows:

Scavenging outcome% = [1- (A₁-A₂)/A₀] * 100

A0 represents the absorbance of the control, which is water rather than the sample; A1 represents the absorbance of the sample; and A2 represents the absorbance of the sample solely, which is phosphate buffer rather than H₂O₂ solution. It was the concentration of the chemicals that generated a 50% inhibition of H₂O₂ that was represented by the IC50 value¹⁴.

III. Nitric oxide radical scavenging activity

Griess reagent was utilized in order to ascertain the concentration of nitric oxide scavenging activity. After preparing 5mM of sodium nitroprusside in phosphate buffer saline (PBS), the mixture was mixed with 3.0ml of extract that had different dilutions ranging from 20–320μg/ml. Following this, the mixture was incubated at a temperature of 25°C for a duration of 150 minutes. Following this, the mixture was analyzed. The samples were then loaded with 5milliliters of Griess reagent, which consisted of 1% sulphanilamide, 2% hydrogen peroxide, and 0.1% naphthalene-diamine dihydrochloride. The absorbance was measured at 546 nm, and it was compared to the absorbance of standard solutions of ascorbic acid that had been treated in the same manner with Griess reagent as a positive control.

The percentage of inhibition was measured by the following formula:

% Inhibition = [(A₀-A_T)/A₀ x100]

In this equation, A0 represents the absorbance of the control, which is a blank sample that does not contain any extract, and At represents the absorbance when the extract is present. A graph was created using the mean data, and each of the experiments was carried out three times to ensure accuracy¹^5,16.

IV. TAOC

The TAC was analysed using a modified version of the phosphomolybdate method that was developed by (Prieto et al. 1999). In the beginning, a plant sample that was 0.3milliliters in volume and contained 1 milligramme per milliliter was mixed with 3milliliters of phosphomolybdate and heated to 95 degrees Celsius for 10minutes. This was done in order to determine the concentration of the plant. Therefore, the absorbance was measured at a wavelength of 695nm. The ascorbic acid calibration curve, which ranged from 10 to 320 g/mL, was then utilized to determine the TAC, which was defined as mg AAE/g of the sample's raw weight.

V. Determination of DPPH (1-1-diphenyl 2-picryl hydrazyl) radical scavenging activity:

An initial step involved the preparation of a 0.1mM DPPH solution in ethanol. Following this, 1.0ml of the DPPH solution was added to 3.0ml of extract solution in water, with the concentrations ranging from 20 to 320 μg/ml. Ascorbic acid was used as the reference compound, and the absorbance was measured at 517nm after thirty minutes elapsed. Free radical scavenging activity is increased when the absorbance value is decreased. The percentage of free radicals that were inhibited by the sample was used to express the free radical scavenging activity, and the formula that was used to calculate it is as follows:

% Inhibition= [(A₀-A_t)/A₀ x 100]

In this equation, the absorbance A0 represents the absorbance when the extract is not present (the blank), and the absorbance At represents the absorbance when the extract is present. There were three separate sets of tests that were carried out, and the graph was constructed using the average values¹⁷.

VI. Estimation of total phenolic compounds:

The Folin-Ciocalteau reagent was utilized for the estimation process, and gallic acid was utilized as the industry standard for phenolic compounds. An extract solution with a concentration of 1mg/mL was removed from a volumetric flask in accordance with the given procedure. The completion of the reaction mixture was accomplished by combining 0.5millilitres of plant extract solution with 2.5millilitres of a 10% Folin Ciocalteu reagent that had been dissolved in water and 2.5millilitres of an aqueous solution of 7.5% sodium hydroxide. The samples were kept at 45 degrees Celsius for a period of forty-five minutes. At a wavelength of 760nm, the absorbance values of the blue color were measured. The amount of total phenols was expressed as milligrams per gram of dry extract throughout the experiment. There were three separate sets of results for each determination. The amount of gallic acid equivalents (GAE) needed to calculate the total amount of phenolic compounds present in the plant extract was measured in milligrams.

LC-MS analysis:

The LC-MS technique was employed to identify and analyze phytochemicals present in CIAQ (Cissampelos pareira aqueous extract) and CIME (Cissampelos pareira methyl extract). The sample were prepared by reconstituting 100mg/mL dried extracts. The sample was then homogenized using mortar pestle and centrifuged at 12,000rpm for 5minutes at 4°C. The supernatant was collected and filtered through a 0.45µm syringe filter. The filtered sample were then subjected to further analysis. The processed samples were analysed through reverse phase-liquid chromatography that employed an Agilent 6470 LC-MS TQ series system. The samples injection volume was set to 10µL using an auto-sampler. After that, the mobile phase, composed of 5mM ammonium formate in 0.1% formic acid (prepared in LC-MS grade water), was delivered at a flow rate of 0.4 mL/minute for the first 20minutes. This was followed by a methanol gradient flow of 5, 30 and 100% for 3, 17 and 20minutes, respectively, with an additional post-run of 3minutes. The phytochemicals in the extracts were detected within 20 minutes. The obtained chromatographic data were analyzed using MASSHUNTER software. The phytochemicals were quantitatively and qualitatively analyzed by comparing the mass spectra of different extracts to those in the NIST library (NIST08 Mass Spectral Search Programme, Gaithersburg, MD, USA).

RESULTS:

Phytochemical screening

Quantitative phytochemical screening:

The amount of flavonoids, saponins and terpenoid was found to be 11.58% w/w, 13.56% w/w, 0.056% w/w respectively.

Qualitative phytochemical screening

Several bioactive components, including saponin, alkaloid, terpenoid, steroid, glycoside, flavonoids, tannins, proteins, carbohydrates, amino acids, and fixed oils, were found during the preliminary phytochemical screening of the Cissampelos pareira extracts in this work.

Table 1 Qualitative analysis of various extracts of Cissampelos pareira

S. No	Phytoconstituents	*Cissampelos pareira*
S. No	Phytoconstituents	Aqueous	Methanol
1	Saponin	+	+
2	Alkaloid	-	-
3	Terpenoid	-	-
4	Steroid	-	-
5	Glycosides	-	+
6	Flavonoids	+	+
7	Tannins	+	+
8	Proteins	-	-
9	Carbohydrates	+	+
10	Amino acids	+	-
11	Fixed oils	-	-

Determination of antioxidant activity:

The DPPH radical scavenging activity results showed that both extracts were effective. Alternatively, it was discovered that ascorbic acid exhibited a notable inhibitory impact between 10µg/ml and 320µg/ml. Maximum inhibition effect was observed CIAQ (66.25%), (72.40%) in CIME extract and (82.51%) in Ascorbic acid.

The IC50 value were found to be CIAQ (189.68μg/ml), CIME (179.5μg/ml), while that of ascorbic acid was found to be 121.73μg/ml. The results indicate that both plant extracts have DPPH scavenging activity that is on par with that of the gold standard, ascorbic acid (Figure 1).

Reducing power activity

The reducing power of CIAQ, CIME extracts were assessed at different concentrations (10μg/ml to 320μg/ml). The reducing power of CIAQ, CIME extracts of was found to be increased with concentration. At 320μg/ml, the extracts exhibited the maximum reducing power. EC50 was found to be 29.22µg/ml with CIAQµg/ml and 33.76µg/ml CIME extract. The value for ascorbic acid was 14.72µg/ml (Figure 1).

Hydrogen peroxide-scavenging activity

It was discovered that the CIAQ, and CIME extracts inhibited hydroxyl radicals to the maximum percentage of 67.739% and 69.837% specifically, at a concentration of 320μg/ml. In contrast, ascorbic acid showed 77.7% inhibition. The ability of the extracts to scavenge these radicals was shown to be concentration dependent. The IC50 value of CIAQ, CIME extracts was found to be 194.55μg/ml and 183.87μg/ml. The ascorbic acid IC50 value was determined to be 148.71μg/ml (Figure 1).

Total antioxidant capacity (TAC)

When measured against an ascorbic acid reference standard CIAQ, CIME displayed potential antioxidant ability, that is 148.78, 178.04mg/AAE (Ascorbic acid equivalent), respectively (Figure 1).

Nitric oxide radical scavenging activity

The Griess reagent was employed to determine the nitric oxide scavenging activity. To begin, 5millimolar of sodium nitroprusside was prepared in PBS. The next step was to add 3.0mL of extract, which had a concentration of 20–320mg/mL. The combination was then incubated at 25°C for 150 minutes. In NO scavenging activity, IC50 value of CIAQ 200.92, CIME 201.98μg/ml while that of ascorbic acid was found to be 152.97μg/ml (Figure 1).

Total phenol content

The total phenol content of CIAQ, CIME extracts was found to be 503.4, 522.4mg/Gallic acid equivalent (GAE), respectively (Figure 1).

LC-MS analysis:

LC-MS techniques were employed to determine the prominent compounds present in different extracts. Major compounds like Limonin, Hercynine, Costunolide, Daidzein, Chrysin were found in CIAQ and CIME extracts and trace amounts of other phytochemicals were identified by the LC-MS analysis (Figure 2).

Figure 2 Representative chromatogram indicating chemical profile of CIAQ and CIME as determined by LC-MS.

DISCUSSION:

The qualitative phytochemical analysis for the plant showed extracts showed noticeable levels of certain bioactive compounds like flavonoids (11.58% w/w) and saponins (13.56% w/w), while traces of terpenoids (0.056% w/w) were also found. In the DPPH radical scavenging test, both CIAQ and CIME extracts showed a dose-dependent antioxidant activity, having maximum inhibition of 66.25% for CIAQ and 72.40% for CIME, while ascorbic acid inhibited it by 82.51%, with IC₅₀ values of 189.68µg/ml (CIAQ), 179.5µg/ml (CIME), and 121.73µg/ml (ascorbic acid), indicating that the extracts demonstrated remarkable scavenging potential, especially the methanolic extract. Likewise, reducing power activity increased with concentration, showing maximum effect at 320µg/ml, with EC₅₀ values of 29.22 µg/ml (CIAQ) and 33.76µg/ml (CIME), compared to 14.72µg/ml for ascorbic acid. Hydrogen peroxide scavenging activities also revealed marked inhibition. The CIAQ and CIME showed maximum inhibition of 67.739 and 69.837%, respectively at 320µg/ml, while ascorbic acid inhibited 77.7%. Their IC₅₀ values were estimated to be 194.55 µg/ml for CIAQ, 183.87µg/ml for CIME, and 148.71µg/ml for ascorbic acid, supporting the ability of the extracts to neutralize ROS. The total antioxidant capacity (TAC) assay also confirmed the strong antioxidant potential of the compounds as both CIAQ and CIME revealed 148.78 and 178.04mg AAE, respectively. The nitric oxide radical scavenging activity of the compounds was found to be moderate, and the values for both CIAQ and CIME were 200.92 and 201.98µg/ml, respectively, as compared to 152.97µg/ml for ascorbic acid. The total phenolic content is notably high in both CIAQ and CIME, containing 503.4 and 522.4mg GAE, respectively. Furthermore, using the LC-MS chromatogram, compounds like Limonin, Hercynine, Costunolide, Daidzein, Chrysin were found in CIAQ with concentration 205.09ng/ml, 480.66ng/ml, 244.85 ng/ml, 326.30ng/ml, 261.72ng/ml, respectively and in CIME extracts concentration was 199.67 ng/ml, 467.94 ng/ml, 238.37ng/ml, 422.21 ng/ml, 338.64 ng/ml, respectively.

CONCLUSION:

The present study, therefore, concludes that Cissampelos pareira possesses a high level of phytochemical content and a tremendous amount of antioxidant activity. The results obtained in the present study by conducting various phytochemical tests proved the existence of high amounts of major bioactive chemical compounds such as flavonoids, saponins, tannins, carbohydrates, and glycosides in Cissampelos pareira. The results obtained in the present study also proved the high antioxidant activities of aqueous and methanolic extracts of Cissampelos pareira, which were found to possess a high percentage of DPPH free radicals, reducing power, and hydrogen peroxide-scavenging activities in a dose-response manner. Major bioactive chemical constituents, namely Limonin, Hercynine, Costunolide, Daidzein and Chrysin, were also identified by LC-MS, which are very well known for their antioxidant and protective activities. Overall, the results confirm the potential therapeutic value of Cissampelos pareira for the development of antioxidant agents, scientifically validating the traditional medicinal use of the species with antioxidant activities. It is recommended that further research be undertaken in order to validate the therapeutic application in the treatment of oxidative stress-related diseases.

REFERENCES:

1. Reddy, G.D., C.V. Rao, and A. Shirwaikar. Ethnomedical value of Cissampelos pareira extract in experimentally induced diarrhoea. Acta Pharmaceutica. 2004: 54(1): 27-35.

2. Kumari, S., et al. Cissampelos pareira L.: a review of its traditional uses, phytochemistry, and pharmacology. Journal of Ethnopharmacology. 2021: 274: 113850.

3. Ngoci, N.S., et al. Screening for antimicrobial activity of Cissampelos pareira L. methanol root extract. European Journal of Medicinal Plants. 2014; 4(1): 45-51.

4. Lalit, K., et al. Phyto-pharmacological review of Coccinia indica. World Journal of Pharmacy and Pharmaceutical Sciences. 2014; 3(2): 1734-1745.

5. Kabilan, S.J., et al. Therapeutic efficacy of polyherbal formulation kabasura kudineer against common viral fevers-a perspective review. Anti-Infective Agents. 2022; 20(5): 63-80.

6. Thakur, S., H. Kaurav, and G. Chaudhary. Karanj (Pongamia Pinnata)–an ayurvedic and modern overview. Asian J Pharm Clin Res. 2021; 14(6): 14-21.

7. Gul, M.Z., et al. Antioxidant and enzyme inhibitory activities of Cissampelos pareira L. leaf extracts. Ann. Phytomed. 2016. 5(1): 91-8.

8. Shaheedha, S., T. AK, and N. Parveen. Phytochemical Screening and Evaluation of Wound Healing Activity on Roots of Achyranthes Aspera Linn. International Journal of Pharma Research. 2023; 13(2).

9. Odey, M., et al. Preparation of plant extracts from indigenous medicinal plants. International Journal of Science and Technology. 2012; 1(12): 688-692.

10. Uthayarasa, K., et al. Antibacterial activity and qualitative phytochemical analysis of medicinal plant extracts obtained by sequential extraction method. IJIB. 2010; 10(2): 76-81.

11. Shaikh, J.R. and M. Patil. Qualitative tests for preliminary phytochemical screening: An overview. International Journal of Chemical Studies. 2020; 8(2): 603-608.

12. Santhi, K. and R. Sengottuvel. Qualitative and quantitative phytochemical analysis of Moringa concanensis Nimmo. International Journal of Current Microbiology and Applied Sciences. 2016; 5(1): 633-640.

13. Yen, G.C. and P.P. Hsieh. Antioxidative activity and scavenging effects on active oxygen of xylose‐lysine Maillard reaction products. Journal of the Science of Food and Agriculture. 1995; 67(3): 415-420.

14. Ruch, R.J., S.J. Cheng, and J.E. Klaunig. Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis. 1989; 10(6): 1003-1008.

15. Shukla, S., et al. In vitro antioxidant activity and total phenolic content of ethanolic leaf extract of Stevia rebaudiana Bert. Food and Chemical Toxicology. 2009; 47(9): 2338-2343.

16. Marcocci, L., et al. The nitric oxide-scavenging properties of Ginkgo biloba extract EGb 761. Biochemical and Biophysical Research Communications. 1994; 201(2): 748-755.

17. Blois, M.S. Antioxidant determinations by the use of a stable free radical. Nature. 1958; 181(4617): 1199-1200

Received on 11.12.2025 Revised on 24.02.2026

Accepted on 25.04.2026 Published on 20.05.2026

Available online from May 25, 2026

Research J. Pharmacy and Technology. 2026;19(5):2223-2228.

DOI: 10.52711/0974-360X.2026.00320

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.