Abstract: Background and Objective: Various parts of medicinal plants have been used to treat specific disorder from ancient times. Swertia chirayita (Roxb. ex Fleming) is a customary folklore medicine, used in the treatment of liver disorders, fevers, dysentery, diarrhea, stomach problems and other disorders. The present study was carried out in order to assess the antioxidant activity, to evaluate the antifungal properties of the plant’s root and to observe anticancer potential of methanolic extract of Swertia chirayita root. Materials and Methods: Phytochemical analysis and different chemical tests for the screening and identification of bioactive chemical constituents in Swertia chirayita methanolic root extract (SCME) were carried out using the standard procedures. The plants were purchased from local herbal market. In vitro determination of antioxidant properties of SCME were conducted using 1,1-diphenyl-2-picrylhydrazyl (DPPH)radical scavenging activity, H2O2 scavenging activity, Beta-carotene bleaching assay, total antioxidant activity by phosphomolybdenum method, azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation scavenging activity and hydroxyl radical scavenging activity. Anticancer activity of SCME was determined according to the protocol of brine shrimp lethality test. Antifungal potential was determined by measuring zone of inhibition on Potato Dextrose Agar (PDA) plates. The different concentrations of SCME used were 3.0, 1.5, 0.75 and 0.37 mg mL–1. The data were evaluated as Mean±Standard Deviations of 5 independent experimental responses. The results were analyzed using t-test for independent samples with SPSS version 16.0. Results: Phytochemical analysis of SCME showed that phlobatannins, tannins, saponins and terpenoids were present. The SCME exhibited strong antioxidant activity in a concentration dependent manner for in all six models. The SCME at dose of 3 mg mL–1 caused 100% death rate of brine shrimp after 72 h. The SCME showed potent activity against Aspergillus flavus (87%) followed by Aspergillus niger (88%) while the highest activity was shown against Aspergillus fumigatus (92%). Conclusion: The SCME exhibited strong antioxidant, antifungal and cytotoxic potential. Purification of different bioactive compounds should be carried out and in vivo studies are required for further verification.
INTRODUCTION
Medicinal plants are conventionally used worldwide for treatment of different disorders related to human health1. Different preparations of these plants have been found to be greatly effective against various infections caused by micro-organisms. About 70,000 plant species have been used from time to time for curing many microbial diseases1,2. Various parts of medicinal plants have been used to treat specific disorder from ancient times. The indigenous systems of medicine namely Ayurvedic, Unani and Siddha have been used for several centuries. The indigenous systems of medicine used traditionally, fulfill the medical treatment requirements of 70% of village population3. The demands of modern pharmaceutical industries for medicinal plants has also been increased several times2.
An imbalance between the production of free radicals and the endogenous action of antioxidant defense systems results in oxidative stress, which is divided into enzymatic and nonenzymatic systems. Compounds such as β-carotene, α-tocopherol (Vitamin E), sodium ascorbate (Vitamin C) and phenolic compounds which are widely found in medicinal plants contribute to non-enzymatic antioxidant defense systems3-5. The main function of antioxidant defense system is to prevent or reduce the extent of damage caused by free radicals and other reactive oxygen species. These damages may be responsible for a number of chronic diseases, including cancer, some cardiovascular and neurodegenerative diseases6.
At present cancer is a highly lethal disease in Pakistan as well as worldwide. In the treatment of cancer medicinal plants play a crucial role7,1. Swertia chirayita (Roxb. ex Fleming) (S. chirayita) is from gentian family 8,9. It is native to many areas of the northern hemisphere, comprising many regions of Eurasia and western North America9. Swertia (Gentianaceae) is a large genus of plants distributed in the mountainous regions of tropical areas. Most of the species found in India grow at high altitude in the temperate Himalayas from Kashmir to Bhutan and also in the Khasia and Western Ghats hills and Himalayan regions of Pakistan10. Other important species, such as S. angustifolia, S. corymbosa, S. decussata, S. hookeri, S. macrosperma, S. petiolata, S. lawii, S. paniculata, S. punctata, S. calycina, S. purpurascens, S. bimaculata, S. ciliata, S. densifolia, S. japonica and S. frachetiana are also from the customary folklore medicine and are being used as alternatives for S. chirayita in India, China, Pakistan, Japan and other Asian countries in the cure of liver disorders, fever, dysentery, diarrhea, stomach problems and other disorders11.
Both the crude and purified extracts significantly inhibited (p<0.001) cell proliferation and induced apoptosis12. This is the first report of its kind and the observation suggests the chemo-preventive potential of S. chirayita. The ethanolic and methanolic extracts of aerial parts of S. chirayita exhibited anti-cancer activity against a human nasopharyngeal epidermis tumor cell line (KB) whereas in a recent study the S. chirayita showed 100% anticancer activity13. Saha and Das14 reported that the anti cancer activity was observed when the crude extract (10-320 μg mL–1) of the plant was tested against 7, 12-dimethylbenz[a] anthracene (DBMA) induced mouse skin carcinogenesis model. It has been reported that methanol extracts of both leaves and stem exhibited anticancer activity on HCT 116 cell lines in which the methanol extract of leaves exhibited more anticancer activity compared to stem15.
Phenolic compounds are derived from secondary plant metabolism and are essential for plant growth and reproduction. Flavonoids, anthraquinones, xanthones, phenolic acids, saponines, tannins and tocopherols are the most common natural source of phenolic antioxidants. It has been showed in the present study that root of S. chirayita contains phlobatannins, saponines, tannins and terpenoids16,17. They are used for treatment of variety of disorders. In Indian medical system, these plants are used as a remedy for liver disorders, bronchial asthma, chronic fevers, anemia and diarrhea18. In Ayurveda, S. chirayita is used as antipyretic, antihelmintic, laxative and also used in treatment of asthma and leucorrhoea19. In Unani medicine, it is used as astringent, tonic and anti-inflammatory agent. While various parts of S. chirayita are considered as active remedies, the roots are one of the most potent parts. Phytochemical studies have been shown the presence of xanthone derivatives, flavonoids, iridoid glycoside and triterpenoids in this genus20. Because of the presence of different components, various biological activities such as hepato-protective, antimicrobial, anti-inflammatory, anticarcinogenic, hypoglycemic, antimalarial, antioxidant and CNS depressant properties have been reported for these plants21.
The methanolic and aqueous whole plant extracts S. chirayita, at a dose of 200 and 300 mg kg–1, produced significant (p<0.05) hepato-protection as shown by decrease in the activities of the serum enzymes and bilirubin while there were marked scavenging of the DPPH free radicals by the fractions22,23 .The findings of In vitro studies showed that at 25 mg mL–1 (p>0.05) the crude aqueous (CAE) and methanolic extracts (CME) of S. chirayita whole plant displayed an anthelmintic influence on live Haemonchus contortus. Furthermore, when sheep naturally infected with mixed species of gastrointestinal nematodes were treated with whole plant Crude Powder (CP) of S. chirayita, CAE and CME at the dose of 3 g kg–1, exhibited a substantial decrease in egg per gram of feces24.
Methanolic extract showed activity against Cladosporium oxysporum (9 mm at dose of 800 μg mL–1), moderate activity against Aspergillus niger (2 mm at dose of 800 μg mL–1) and no activity against Aspergillus flavus25. Dichloromethane fraction from both leaf and stem of S. chirayita showed significant antimicrobial activities against some gram-positive and gram-negative bacteria and mild to moderate activity against some fungi26. Growth of Candida tropicalis was inhibited by S. chirayita extracts. The significant MIC value was 10 mg mL–1 against Candida tropicalis2. Methanol extract of S. chirayita showed only moderate antifungal activity against A. niger and Penicillium27. Herbal medicines are considered comparatively harmless and have been used in the management of liver maladies for a long time. The effectiveness of herbal medicines needs to be appraised through carefully designed multi center clinical studies28. The findings of this study about various pharmacological properties of SCME will form the basis of research study with purified phytochemicals present in SCME.
The present study was designed to carry out phytochemical analysis and to evaluate different pharmacological effects of crude extract of root S. chirayita.
MATERIALS AND METHODS
Chemicals: Ferric chloride, 2,2-diphenyl-1-picryl-hydrazyl, potassium persulfate, azinobis(3-ethylbenzothiozoline-6-sulfonic acid) disodium salt, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, ferrous chloride, hydrogen peroxide, ferrous ammonium sulfate, ethylenediaminetetracetic acid (EDTA) disodium salt were obtained from Hi-media, Mumbai, India. 2,2’-bipyridyl and hydroxylamine hydrochloride, Penicillin G and streptomycin [3,-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-tetrazolium bromide] (MTT) were purchased from Sigma (St. Louis, USA). Fetal bovine serum, Dulbecco’s modified Eagle medium were purchased from Biochrom, Berlin, Germany. All other chemicals and reagents used were of analytical grade.
Plant collection: The roots of S. chirayita were purchased from local herbal market of Bannu, Pakistan, shade dried and grounded to powder which was saturated with methanol to prepare methanolic crude extract.
Extract preparation: The root powder (60 g) of S. chirayita was soaked in 20 mL of 80% methanol, continuously shaken for one hour and then kept at room temperature for seven days. Whatman No. 1 filter paper was used to filter the plant extract after seven days. The filtrate was further concentrated and dried on rotary evaporator under reduced pressure at 40°C, then it was stored in refrigerator at 4°C for further use.
Each sample was dissolved in 95% methanol at a concentration of 3 mg mL–1 and then diluted to prepare the series of concentrations for different assays.
In vitro determination of antioxidant activity: Antioxidant property of plant samples was carried out according to the method described by Wojdylo et al.29. Samples (1 g) were added to 10 mL of 80% methanol and the suspensions were stirred slightly. Tubes were vortexed twice for 15 min and left at room temperature (25°C) for 24 h. The extracts were centrifuged for 10 min at 1500 rpm and supernatants were kept at 4°C prior to use within 24 h.
DPPH radical scavenging activity: The 1,1-diphenyl-2- picrylhydrazyl (DPPH) activity was assessed by 1,1-diphenyl-2- picrylhydrazyl (DPPH) radical scavenging assay according to the methods described by Shimada et al.30. DPPH solution (0.1 mM) was freshly prepared in ethanol and was used for the assay. Then, 1 mL of this solution was added to 2 mL of SCME solution at different doses of 3.0, 1.5, 0.75 and 0.37 mg mL–1. The mixture was shaken vigorously and kept in the dark at controlled room temperature (25-28°C) for 30 min. Then the absorbance was measured at 517 nm. The degree of discoloration occurred in the samples indicates the free radical scavenging efficiency of the substances. The percentage of DPPH radical scavenging activity was calculated by the following formula30:
| (1) |
where, Abr is the absorbance before reaction and Aar is the absorbance after reaction.
Total antioxidant capacity:The total antioxidant capacity of the extracts was evaluated by the phosphomolybdenum method according to the procedure described by Prieto et al.31. A 0.3 mL of 3.0, 1.5, 0.75 and 0.37 mg mL–1 SCME solution was combined with 3 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes containing the reaction solution were incubated at 95°C for 90 min. Then, the absorbance of the solution was measured at 695 nm using a UV-VIS spectrophotometer (UVmini-1240) against blank after cooling to room temperature. Methanol (0.3 mL) in the place of extract was used as the blank. The total antioxidant activity is expressed as the number of gram equivalent of ascorbic acid. The calibration curve was prepared by mixing ascorbic acid (1000, 500, 250, 125, 62.5 and 31.25 μg mL–1) with methanol.
Hydroxyl radical scavenging activity: The scavenging activity of the SCME solution on hydroxyl radical was measured according to the method of Klein et al.32. Various concentrations (3.0, 1.5, 0.75 and 0.37 mg mL–1) of extracts were added with 1 mL of iron-EDTA solution (0.13% ferrous ammonium sulfate and 0.26% EDTA), 0.5 mL of EDTA solution (0.018%) and 1 mL of dimethyl sulfoxide (DMSO) (0.85% v/v in 0.1 M phosphate buffer, pH 7.4). The reaction was initiated by adding 0.5 mL of ascorbic acid (0.22%) and incubated at 80-90°C for 15 min in a water bath. After incubation the reaction was terminated by the addition of 1 mL of ice-cold TCA (17.5% w/v). Three milliliters of Nash reagent (75 g of ammonium acetate, 3.0 mL of glacial acetic acid and 2 mL of acetyl acetone were mixed and raised to 1 L with distilled water) was added and left at room temperature for 15 min. The reaction mixture without sample was used as control. The intensity of the color formed was measured spectroscopically at 412 nm against reagent blank. The Hydroxyl Radical Scavenging Activity (HRSA) was calculated by the Eq. 2:
| (2) |
where, A0 is the absorbance of the control and A1 is the absorbance of the extract/standard.
Scavenging of H2O2: Scavenging of H2O2was determined by the method reported by Alam et al.33. About 4 mM H2O2, 0.6 mL solution (prepared in PBS) was added to 4 mL solution of extract containing 3.0, 1.5, 0.75 and 0.37 mg mL–1 and incubated for 10 min. The absorbance of the solution was measured at 230 nm by using spectrophotometer. Free radical scavenging potency as determined from percentage of age H2O2. Lower percentage of age H2O2 indicated strong free radical scavenging activity. Percent scavenging of the DPPH free radical was measured using the Eq. 334.
| (3) |
where, Ac is absorbance of control and As is absorbance of sample solution.
ABTS radical cation scavenging activity: Antioxidant activity of this plant was measured using an improved ABTS method as described by Re et al.35. The ABTS•+ was produced by reacting 7 mM ABTS aqueous solution with 2.4 mM potassium persulfate in the dark for 12-16 h at room temperature. Prior to assay, this solution was diluted in ethanol (about 1:89 v/v) and equilibrated at 30°C to give an absorbance of 0.70±0.02 at 734 nm. The stock solution of the sample extracts were diluted such that after introduction of 10 μL aliquots into the assay, they produced between 20 and 80% inhibition of the blank absorbance. After the addition of 1mL of diluted ABTS solution to 10 μL of sample or Trolox (final concentration 0-15 μM) in ethanol, absorbance was measured at 30°C exactly 30 min after the initial mixing. Triplicate determinations were made at each dilution of the standard and the percentage inhibition was calculated against the blank (ethanol) absorbance at 734 nm and then was plotted as a function of Trolox concentration. The unit of total antioxidant activity is defined as the concentration of trolox having equivalent antioxidant activity expressed as μM g–1 sample extracts. The extract at different concentrations (3.0, 1.5, 0.75 and 0.37 mg mL–1) were used to assess the antioxidant activity.
β-carotene bleaching assay: β-carotene (10 mg) was dissolved in 10 mL of chloroform. The carotene-chloroform solution (0.2 mL) was pipetted into a flask containing 20 mg of linoleic acid and 200 mg of Tween 40. Chloroform was removed using a rotary evaporator under reduced pressure at 40°C for 5 min and 50 mL of distilled water was added slowly to the residue with vigorous agitation in order to form an emulsion. A 4.8 mL aliquot of the emulsion was added to a tube containing 0.2 mL of the sample solution and the absorbance at 470 nm was immediately measured against a blank that was an emulsion without β-carotene. The tubes were placed in a water bath at 50°C and the oxidation of the emulsion was monitored spectrophotometrically by measuring the absorbance at 470 nm for 60 min. Control samples contained 0.2 mL of ethanol instead of the sample extracts. Ascorbic acid was used as the reference. The antioxidant activity was expressed as percent inhibition with reference to the control after 60 min of incubation using the following formula36:
| (4) |
where, AA is the antioxidant activity, DRC is the degradation rate of the control {DRC = [ln(a/b)/60]}, DRS is the degradation rate in the presence of the sample {DRS = [ln(a/b)/60]}, where a is the absorbance at time 0 and b is the absorbance after 60 min36. The antioxidant activity of SCME was determined using different concentrations i.e. 3.0, 1.5, 0.75 and 0.37 mg mL–1 of SCME solution.
Cytotoxic activity assay: The cytotoxic brine shrimp lethality test was done by following procedure of Meyer-Alber et al.37. Brine shrimp lethality bioassay is a recent development in the bioassay for bioactive compounds, which indicates cytotoxicity as well as a wide range of pharmacological activities (e.g., anticancer, antiviral, pesticidal etc.) of the compounds. Bioactive compounds are almost always toxic in high doses. Here in vivo lethality of a simple zoological organism (brine shrimp nauplii) is used as a convenient monitor for screening of new bioactive natural products. Generally the median Effective Dose (ED50) values for cytotoxicities are one tenth (1/10) of median Lethal Dose (LC50) values in the brine shrimp test. The anticancer activity (cytotoxicity) of SCME was determined against brine shrimps growth using different concentrations i.e. 3.0, 1.5, 0.75 and 0.37 (mg mL–1) of SCME solution.
Determination of antifungal activity: Method of Duraipandiyan and Ignacimuthu38 was followed to assess antifungal activity of S. chyrayita methanolic extract (SCME). Similarly for the antifungal activity, the stock cultures of fungi (Aspergillus niger (MTCC 1881), Aspergillus flavus (MTCC 1883), Cladosporium oxysporum (MTCC 1777), were revived by inoculating in broth media and grown at 27°C for 72 h. The agar plates of the Potato Dextrose Agar (PDA) media were prepared. Each plate was inoculated with an aliquot (0.1 mL) of the fungal suspension (103 spores mL–1), which was spread evenly on the plate. After 20 min, wells were made and filled with test samples of different concentrations. The positive control plates with Terbinofine (standard drug) and negative control plates with DMSO were also prepared. All the plates were incubated at 27°C for 72 h and then the diameter of zone of inhibition was noted. The antifungal activity of SCME was determined against above mentioned 3 different fungal strains using different concentrations i.e. 3.0, 1.5, 0.75 and 0.37 mg mL–1 of SCME solution.
Preliminary phytochemical screening: Chemical tests were carried out on the methanolic extract to identify the constituents using standard procedures as described by Harborne39, Sofowora40 and Trease and Evans41.
Test for tannins: About 2 mL of them ethanolic extract was stirred with 2 mL of distilled water and few drops of FeCl3 solution were added. The formation of a green precipitate was an indication for the presence of tannins.
Test for saponins: About 5 mL of methanolic extract was shaken vigorously with 5 mL of distilled water in a test tube and warmed. The formation of stable foam was taken as an indication for the presence of saponins.
Test for phlobatannins: About 2 mL of methanolic extract was added to 2 mL of 1% HCl and the mixture was boiled. Deposition of a red precipitate was taken as an evidence for the presence of phlobatannins.
Test for terpenoids: About 2 mL of the methanolic extract was dissolved in 2 mL of chloroform and evaporated to dryness. Then 2 mL of concentrated sulphuric acid was added and heated for about 2 min. A greyish color indicates the presence of terpenoids.
Statistical analysis: The data were evaluated as Mean±Standard Deviations of five independent experimental responses. The results were analyzed using t-test for independent samples with SPSS version 16.0. The significance of the results is statistically satisfactory with significance level less than 0.1%.
RESULTS
In vitro antioxidant activities
DPPH radical scavenging activity: The DPPH radical scavenging activity of SCME was assessed at different concentrations 3.0, 1.5, 0.75 and 0.37 mg mL–1. It was observed that SCME was capable of neutralizing the DPPH free radicals via hydrogen donating activity in a dose dependent manner (Table 1). The maximum DPPH radical scavenging activity of SCME was recorded at a dose of 3.0 mg mL–1 which was 88.1±0.1%.
Total antioxidant capacity: The total antioxidant capacity of SCME was assessed at different concentrations 3.0, 1.5, 0.75 and 0.37 (mg mL–1). Maximum activity; 92±0.2% was found in 3.0 mg mL–1 of SCME extract (Table 2).
Hydroxyl radical scavenging activity: The scavenging activity of the SCME solution on hydroxyl radical was measuredat various concentrations 3.0, 1.5, 0.75 and 0.37 (mg mL–1). The concentration of 3.0 mg mL–1 of SCME extract exhibited maximum activity; 84.33±0.2% (Table 3).
| Table 1: | Scavenging activities of ascorbic acid and SCME for DPPH free radicals |
| SEM: Swertia chirayita methanolic root extract, Data are presented as Mean±SD | |
| Table 2: | Scavenging activities of ascorbic acid and SCME against phosphomolybdate free radical |
| Data are presented as Mean±SD | |
| Table 3: | Scavenging activities of ascorbic acid and SCME against OH free radicals |
| Data are presented Mean±SD | |
| Table 4: | Scavenging activities of ascorbic acid and SCME against H2O2 free radicals |
| Data are presented as Mean±SD | |
| Table 5: | Scavenging activities of ascorbic acid and SCME against ABTS free radicals |
| Data are presented as Mean±SD | |
| Table 6: | Scavenging activities of ascorbic acid and SCME against β-carotene free radicals |
| Data are presented as Mean±SD | |
Scavenging of H2O2: Scavenging activity of the SCME solution againstH2O2 free radicle was determined at various concentrations i.e. 3.0, 1.5, 0.75 and 0.37 (mg mL–1). The maximum activity; 80.33±0.2% was shown by 3.0 mg mL–1 (Table 4).
ABTS radical cation scavenging activity: Scavenging activities of SCME against ABTS free radicals were estimated using different Concentrations i.e. 3.0, 1.5, 0.75 and 0.37 (mg mL–1). The 3.0 mg mL–1 demonstrated maximum activity; 80.88±0.1% (Table 5).
β-carotene bleaching assay: Scavenging activities of SCME against beta carotene free radicals were determined at various concentrations i.e. 3.0, 1.5, 0.75 and 0.37 (mg mL–1). The maximum activity; 87.03±0.3% was shown by 3.0 mg mL–1 (Table 6).
Cytotoxic activity: The anticancer activity (cytotoxicity) of SCME was determined against brine shrimps growth using different concentrations i.e. 3.0, 1.5, 0.75 and 0.37 (mg mL–1) of SCME solution. It was observed that at concentration of 0.37, 0.75, 1.5 and 3 mg mL–1, 30, 20, 10 and 0% survival and 70, 80, 90 and 100% death occurred, respectively (Table 7).
| Table 7: | Survival and death rate against SCME of brine shrimps |
| Data are mean values of triplicate experiments | |
| Table 8: | Antifungal activity of SCME (Inhibition %) |
| Data are presented as Mean±SD | |
Antifungal activity: The antifungal potential of SCME is highly significant statistically as indicated by p-value (p = 0.000), against fungal strain Aspergillus niger, Aspergillus flavus and Aspergillus fumigates (Table 8). The percentage inhibition of SCME is 90, 89 and 94%, respectively at high 3 mg mL–1 concentration. Negative and positive controls are parts of a well-designed scientific experiment. The negative control group is a group in which no response is expected and positive control, in which a known response is expected. When both are successful, are usually sufficient to eliminate most potential confounding variables, it means that the experiment produces a negative result when a negative result is expected and a positive result when a positive result is expected. Terbinafine was positive control which shows 100% inhibition against these fungal strains, while the DMSO was negative control which shows no inhibition (Table 8).
Phytochemical screening: The phytochemical screening of SCME was also performed with the aim to investigate the presence of various bioactive compounds. This investigation showed the presence of tannins, saponins, phlobatannins and terpenoids in SCME.
DISCUSSION
Free radicals which are produced inside our body during different reactions are scavenged by naturally produced antioxidants and prevent us from the serious effect of tissue or cell damage caused by oxidants. The crude methanolic extract of SC was found highly reactive against DPPH as it showed 88% inhibition by 3.0 mg mL–1. Mahmood et al.23 reported significant scavenging activity against DPPH (51%) by very low dose (35 μg mL–1) of crude extract of whole plant of S. Chirayita. Methanol extract of S. Corymbosa exhibited the highest activity in DPPH radical scavenging activity as mentioned by Mahendran and Bai42. It has been shown that the reducing power and scavenging effects on H2O2, increases sharply with the increasing concentration of the samples and standards to certain extent23. Methanolic root extracts of S. chirayita found to have potential antioxidant activity (56%)43.
The acetone: water (8:2) extract showed the highest Total Phenolic Content (TPC) and DPPH radical-scavenging activity as reported by Singh et al.44. Nair et al.45 reported that a hydro-alcoholic extract of S. chirayita showed highly significant ABTS and DPPH radical-scavenging activity (98 and 93%), respectively. Kshirsagar et al.22 stated that S. chirayita showed better antioxidant activity than other species with highest content of phenolic and flavonoids.
The cytotoxic activity assay of SCME showed a maximum death rate against brine shrimps at 3 mg mL–1 concentration which supports the presence of bioactive element against cancer causing pathogen. It was reported that methanol extracts (10-320 μg mL–1) of both leaves and stem exhibited anticancer activity on HCT 116 cell lines in which the methanol extract of leaves exhibited more anticancer activity compared to stem15. Both the crude and purified extracts significantly inhibited cell proliferation and induced apoptosis as observed by Saha et al.12. The ethanolic and methanolic extracts of aerial parts of S. chirayita exhibited anti-cancer activity against a human nasopharyngeal epidermis tumor cell line (KB) whereas in another study the S. chirayita showed 100% anticancer activity13. Prosenjit and Sukta reported that the anticancer activity was observed when the crude extract (10-320 μg mL–1) of the plant was tested against DBMA induced mouse skin carcinogenesis model14. Shrestha et al.46 reported that the brine shrimp bioassay of methanol and ethyl acetate extract of S. chirayita showed cytotoxicity. The extract showed LC50 of 128.82 ppm. Purified fraction (AJ-1) from rectified spirit extract of S. chirayita also showed significant activity against the brine shrimp (LC50 value of 9.34 μg mL–1), in which the mortality rate increased with the increasing concentration of the compound, suggesting a positive correlation between brine shrimp toxicity and cytotoxicity47.
The SCME exhibited a highly significant antifungal activity against fungal strain Aspergillus niger, Aspergillus flavus and Aspergillus fumigatus. The percent inhibition of SCME was 90, 89 and 94%, respectively at a high 3 mg mL–1 concentration. Laxmi et al.25 revealed that methanolic extract showed significant activity against Cladosporium oxysporum (9 mm at 800 μg mL–1), moderate activity against Aspergillus niger (2 mm at 800 μg mL–1) and no activity against Aspergillus flavus. Candida tropicalis was inhibited by S. chirayita extracts. The significant MIC value was 10 mg mL–1 against Candida tropicalis26. Methanol extract of S. chirayita showed only moderate antifungal activity against A. niger andPenicillium27. Methanolic extract of S. chirayita exhibited significant antifungal with the zone of inhibition comparable to amphotericin46. Only methanolic extract was used in the study, if extract with other solvent e.g. ethanol, acetone and water would have been used, the comparative results were obtained.
CONCLUSION
Thus it is concluded that the various valuable biological activities of the SCME may be due to the presence of phlobatannins, tannins, saponins and terpenoids. Therefore, further analysis of the methanolic crude root extract of S. chirayita for isolation and characterization of bioactive compounds is indispensable.
SIGNIFICANCE STATEMENT
The Swertia chirayita methanolic root extract showed an excellent potential as a therapeutic agent for cancer and other infectious diseases due to the potent antioxidant, antifungal and ant-carcinogenic properties as shown in this study.
ACKNOWLEDGMENT
Authors are thankful to the chairman Department of Biotechnology for providing basic laboratory facilities, guidance and insightful comments during the research study.