The leaves of the fern C. acuminata, Genus: Christella; Family: Thelypteridaceae, were collected from the local surroundings of Majhitar, East Sikkim, India. The plant was identified and authenticated (authentication no. 2706220009379) by the Botanical Survey of India, Sikkim Himalayan Regional Centre, Gangtok. The leaves were then washed to remove fine impurities. The leaves were shade-dried for 4-5 days to remove moisture content and to preserve the maximum bioactive constituents. The dried leaves were further cut down into small pieces and then ground using a grinder to get the powder form. Then the powder was preserved in a well-closed container.

The aqueous and ethanolic extracts were prepared by maceration for 5-7 days. 160g of coarsely powdered C. acuminata was placed in the conical flasks at 1:20 for aqueous extract and at 1:10 for ethanolic extract, respectively. It was then kept for extraction with consecutive shaking for 5-7 days, followed by filtration using a Whatman filter paper. The filtrates were concentrated by evaporating the solvents. The two different concentrated filtrates as a residue were used for anti-inflammatory, antioxidant, and antibacterial activity.

Preliminary phytochemical screening for the presence of alkaloids, saponins, glycosides, polyphenols and tannins, steroids, terpenoids, and flavonoids was carried out as per standard methods. 12, 13

The antibacterial test was conducted in a laminar airflow cabinet using the disc diffusion method. The tests were done in triplicate with sterile petri plates, which were prepared with nutrient agar media. 1 ml of diluted culture was uniformly poured on each Petriplate and kept for 30 minutes at room temperature. Nanoparticles (discs) were placed on the plates, and further, the antibacterial compounds were deposited on each disc. Then the Petri plates were incubated for 24 hours. The efficacy of antibacterial compounds was observed as a zone of inhibition of bacteria around the discs. 14, 15

Serial dilutions from 100µg/ml to 1000µg/ml were prepared for C. acuminata leaf extracts and reference drug (L-ascorbic acid). 2.5 ml of phosphate buffer (0.2M, pH 6.6) was added to all test tubes. Now add 2.5 ml of 1% potassium ferricyanide to each test tube. Then the sample is mixed properly using a vortexed shaker. Achieve incubation for 20 minutes at an optimum temperature of 50ºC. After incubation is completed, add 2.5 mL of 10% trichloroacetic acid to all the test tubes. Centrifuge the sample at 3000rpm for 10 min. After centrifugation is completed, take 2.5 ml of supernatant liquid from all the samples and place it in a test tube with proper labelling. Finally, 2.5 mL of distilled water & 0.5 mL of 0.1% ferric chloride were added to all the test tubes, and absorbance was measured at 700nm using a UV-spectrophotometer and recorded. 16

The anti-inflammatory activity of unknown crude extracts can be determined in vitro for the inhibition of egg albumin (protein denaturation). Serial dilution from 100µg/ml to 1000µg/ml was performed for C. acuminata leaf extracts and reference drug (diclofenac sodium). All samples contained 5.0 ml of total volume. The reaction mixture was prepared using 2.8 ml of phosphate-buffered saline (pH 6.4) and 0.2 ml of 1-2% egg albumin. Then, 2.5 ml of C. acuminata leaf extract from each concentration was mixed gently with the reaction mixtures. A similar procedure was used for the reference drug (diclofenac sodium), and they were used as a positive control for this study. The reaction mixture was incubated in a water bath at 37ºC ± 2ºC for 15-20 min, and later it was heated to 70ºC, at which the reaction mixture was maintained for 5 min. Then, the reaction mixture was allowed to cool down at room temperature for 15 min. The absorbance of the reaction mixture before and after denaturation was measured for each concentration (100µg/ml, 200µg/ml, 400µg/ml, 600µg/ml, 800µg/ml, 1000µg/ml) at 680nm using UV spectrophotometry. Each test was repeated thrice, and the mean absorbance was recorded. The percentage inhibition of protein was determined on a percentage basis compared to the control using the following formula: 7, 17

Percentage inhibition (%) = Absorbanceof Control -Absorbanceof test Absorbanceof Control  x 100

The qualitative phytochemical estimation shows the presence of alkaloids, saponins, polyphenols, tannins, steroids, and flavonoids (Table 1).

To test the antibacterial capacity of the selected ferns, four bacteria, Escherichia coli (Gram negative), Salmonella typhi (Gram negative), Bacillus subtilis (Gram positive) and Staphylococcus aureus (Gram positive) were assessed.

According to the present study, the most potent extract against the above-mentioned bacteria was the ethanol extract compared to the aqueous extract. The antibacterial activity showed that the ethanol extract produced the maximum zone of inhibition compared to the aqueous extract. The inhibition of bacterial (gram +ve and gram –ve) growth by both the extracts of the leaves of C. acuminata (Houtt.) indicated the antibacterial activity of the sample (Table 2).

Figure 2

The antioxidant activity of the leaves of ethanolic and aqueous extracts of C. acuminata was evaluated for their ferric-reducing antioxidant power by the F.R.A.P assay. Results show that all leaves ethanolic and aqueous extracts of the C. acuminata showed the ferric-reducing antioxidant power ranging from 0.188 ± 0.007 to 1.31 ± 0.116 and 0.22 ± 0.005 to 1.8566 ± 0.021 respectively at 700nm by U.V. spectrophotometer. Compared to the different concentrations of standard L-ascorbic acid the aqueous extract exhibits higher ferric-reducing antioxidant power than the ethanolic extract. These observations were carried out in triplicate, and the mean value of absorbance implies that both the ethanolic and aqueous extracts of the fern samples may contain antioxidant molecules that quench the oxidative radicals (Table 3).

When we heat the egg albumin, it generally undergoes denaturation, and antigens are released, which are associated with hypersensitivity reactions related to diseases such as glomerulonephritis, serum sickness, rheumatoid arthritis, and systemic lupus erythematosus.

The results show that both ethanolic and aqueous extracts of the C. acuminata showed the inhibition of the denaturation of albumin in vitro, ranging from 24.77 ± 0.110 to 89.7 ± 0.09 and 18.67 ± 0.420 to 83.973 ± 0.072, respectively. After comparing the results with the standard (Diclofenac sodium) it was found that the ethanolic extract showed better anti-inflammatory activity by percentage inhibition to albumin denaturation (Table 4).

We conducted an investigational study on anti-bacterial, anti-inflammatory and antioxidant activity of fern C. acuminata belonging to the family Thelypteridaceae. The plant was collected from the local area of Majhitar, Rangpo, East Sikkim. The plants were washed and dried in the shade for one week. The process was carried out by the maceration extraction process. Approximately 160g of powdered crude drug of C. acuminata leaves were exhaustively soaked in ethanol and water at 1:10 and 1:20, respectively, for 3-5 days. Occasionally, stirring was done during the soaking by the maceration process. Samples were then filtered using Whatman no.1 filter paper, and the extract was concentrated to a definite volume using a water bath and heating mantle. The test for phytochemicals like Alkaloids, Saponins, Glycosides, Polyphenols & Tannins, Steroids, Terpenoids, and Flavonoids was performed.

The antibacterial activities of different extracts (Aqueous extract and ethanolic extract) were to be tested against Escherichia coli (Gram-negative), Salmonella typhi (Gram-negative), Bacillus subtilis (Gram-positive), and Staphylococcus aureus (Gram-positive). The results showed that all the plant extracts and their solvents gave better inhibition at high concentrations of the extract. According to the present study, the most potent extract against the above-mentioned bacteria was the ethanol extract then followed by the aqueous extract. As a conclusion, the antibacterial activity showed that the ethanol extract produced the maximum zone of inhibition compared to the aqueous extract. Hence, the inhibition of bacterial (gram +ve and gram –ve) growth by both the extracts of the leaves of C. acuminata (Houtt.) shows the antibacterial activity of our sample. During the FRAP test, we prepared different sample concentrations, i.e., 100-1000 µg/ml, using the stock solution. After which, UV Spectroscopy was performed. The absorbance was in increasing order, and the highest absorbance was found for 1000 µl of both ethanolic and aqueous extract; however, the percentage inhibition of Ferric-Reducing Antioxidant Power was found to be highest in the aqueous extract of C. acuminata. During the egg albumin denaturation for anti-inflammatory activity again we prepared different sample concentrations for both ethanolic and aqueous leaf extracts, i.e., from 100-1000 µg/ml using a stock solution, followed by UV spectroscopy. The absorbance was found to be in decreasing order due to the turbidity, and again, the lowest absorbance was found for 1000 µl. The percentage inhibition of denaturation was calculated by comparing the absorbance of the samples and the control, the percentage inhibition was found to be in increasing order with increasing concentration. The highest percentage of inhibition was found in the ethanolic extract of C. acuminata.

An attempt has been made to evaluate the presence of various phytochemicals and antibacterial, antioxidant, and anti-inflammatory properties of leaves of C. acuminata. The identification of the plant material taxonomically and pharmacognostically is important to provide pharmacognostic standards and also to avoid spurious or adulterated drugs. The detailed botanical and pharmacognostical studies help in evolving specific diagnostic characteristics to fulfill this objective.

Although this plant was reputed for its anti-infective properties in traditional medicinal systems, only a limited number of studies have been carried out to evaluate its biological and pharmacological properties. Hence, the present studies have been carried out to evaluate their biological and pharmacological properties. The present study confirmed the occurrence of strong antibacterial, antioxidant, and anti-inflammatory properties in leaf extracts of C. acuminata, which may be due to the presence of polyphenolic compounds such as flavonoids. However, the ethanolic leaf extracts were found to be more effective against both gram-positive and gram-negative bacteria as compared to the aqueous extract. The aqueous extract was found to be more effective for anti-oxidant activity, whereas the ethanolic extract showed better results for anti-inflammatory activity. The findings obtained in this study correspond well with the ethnopharmacological use of C. acuminata for the treatment of bacterial infection, inflammation, and oxidative stress. This study will further pave the way for the isolation and characterisation of useful compounds from this medicinally significant pteridophytic fern. Further in vivo and in vitro studies should be performed to establish the anti-bacterial, antioxidant, and anti-inflammatory activity of C. acuminata leaf.