HPTLC analysis, antioxidant, anti-inflammatory and antiproliferative activities of Arisaema tortuosum tuber extract

Abstract

Context: Oxidative stress and inflammation are related to several chronic diseases including cancer and atherosclerosis. Arisaema tortuosum (Wall.) Schott (Araceae) is an Indian folk medicinal herb traditionally used for treatment of various diseases related to inflammation and stress.

Objective: This study was carried out for HPTLC analysis and evaluation of antioxidant, anti-inflammatory and antiproliferative activities of a methanol extract of A. tortuosum tuber.

Materials and methods: The antioxidant activities of methanol extract of A. tortuosum tuber (1 mg/mL) were evaluated by DPPH, ABTS and FRAP assays and anti-inflammatory effects by diene-conjugate and β-glucuronidase assays, with in vitro tumor growth inhibition on HeLa cancer cells. The results for antioxidant and anti-inflammatory effects were compared using Trolox and salicylic acid as reference compounds, respectively.

Results: The TLC and HPTLC analysis showed the presence of quercetin, rutin, luteolin and lectin (R_f values 0.97, 0.53, 0.59 and 1.58, respectively). The methanol fraction of tuber exhibit higher activity in each antioxidant system with a special attention for DPPH (IC₅₀ = 852 μg/mL), ABTS (IC₅₀ = 532 μg/mL), and FRAP (IC₅₀ = 458 μg/mL), as compared with Trolox as standard, with a remarkable amount of phenolics (86.2 mg/100 g) and flavonoids (175.5 mg/100 g), along with potent anti-inflammatory activity indicated by diene-conjugate (86.20%) and β-glucuronidase (92.92%) inhibition, as compared with salicylic acid as reference compound. The antiproliferative activity at 100 mg/mL was 88% inhibition with HeLa cells. The inhibition of HeLa cell proliferation was greatest (p < 0.001) with the 100 mg/mL A. tortuosum tuber extract treatments and least with the 25 mg/mL dose.

Discussion and conclusion: Our results suggested that A. tortuosum tuber might be used as a promising and potent antioxidant, anti-inflammatory, and antiproliferative agent and might be used for standardization of potential drug after successful isolation and characterization of bioactive compounds.

• Cytotoxicity
• DPPH
• FRAP
• medicinal plant
• phytochemicals
• TLC

Introduction

Arisaema tortuosum (Wall.) Schott (Araceae) (Whipcord Cobra Lily) is a plant species that has a distinctive purple or green whip-like spadix, which arises from the mouth of its “jack-in-the-pulpit” flower and may be up to 30 cm long (Pragada et al., 2012). The plant occurs in rhododendron forest, scrub and alpine meadows in the Himalayas, Western China, Southern India and Myanmar. The dried powder and juice of the tubers was applied to the wounds of cattle in order to kill any parasites and also applied for snake bites (Choudhary et al., 2008; Sharma & Majumdar, 2003). The plant contains calcium oxalate crystals, which cause an extremely unpleasant sensation similar to needles being stuck into the mouth and tongue if they are eaten, but they are easily neutralized by thoroughly drying or cooking. The tubers are buried in masses in pits until acetous fermentation takes place. They are then dug up, washed, and cooked, by which means their acrimonious principles are in part dispersed (Pragada et al., 2012; Punjani, 2006). The tubers have good asthetic, antihepatotoxic, anticancerous, antimicrobial and antioxidant properties (Murty & Narasimha-Rao, 2010). The plant tuber used by Indian tribal people for curing various ailments related to the digestive tract like constipation, indigestion, abdominal pain and dysentery. Chemical research showed that A. tortuosum contained flavonoids, alkaloids, saponins, triterpenoids and lectins (Kamble et al., 2010). Among the protein components, a lectin was proven to be the main pro-inflammatory component, showing anticancer activity against human cancer cell lines (Dhuna et al., 2005). Lectins are (glyco)proteins of non-immune origin that interact reversibly and specifically with carbohydrates which have various biological activities, such as anti-inflammatory, anticancer, immunomodulatory, antifungal, antiviral and anti-insect activity. Moreover, lectins have been shown to present stimulatory effects in different biological models (Swarnkar & Katewa, 2008). These data provide the basis for identifying the basic phytochemicals in A. tortuosum tuber, which could be used for antioxidant, anti-inflammatory and antiproliferative research.

Material and methods

Chemicals

Folin and Ciocalteu’s phenol reagent, sodium carbonate, catechin, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), ABTS, 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), Trolox (6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid), and the HeLa cell line was procured from National Center for Cell Sciences, Pune, India. Cell culture medium components: minimum essential medium (MEM), and phosphate-buffered saline (PBS) were purchased from Bangalore Genei, Bangalore (India). All solvents used were of HPLC grades.

Plant material

Mature whole A. tortuosum tuber was collected from Nanded, Maharashtra, India in October, 2012, identified by Prof. C. N. Khobaragde, Head of Department, Biotechnology and deposited in the herbarium of School of Life Sciences, SRTM, University, Nanded, India. The collected plant tuber was surface sterilized by using 1% mercuric chloride; air-dried in shade, separated, grounded to fine powder and used for further analysis (Pandey & Rizvi, 2009).

Preparation of extracts

For preparation of tuber extracts, 1 kg of tuber sample was extracted with 5 L of methanol overnight at room temperature, filtered and concentrated down to approx. 500 mL by rotary evaporation (Buchi R-200 V, Elico Instruments, Mumbai, India), 40 °C, as previously described (Yuan et al., 2005). After transfer to a separator funnel (1 L volume), the concentrated extract was washed with an equal volume of hexane three times. After separation of the phases at room temperature, the lower methanol phase was extracted with 200 mL H₂O with 300 mL ethyl acetate. The lower H₂O–methanol layer was then extracted with 300 mL 1-butanol in another separator funnel, and the upper butanol layer removed and concentrated by rotary evaporation, 50–55 °C, to obtain the powdered samples. The powdered extracts were solubilized in 25 mM ethanol for further use in assays. The homogenate was kept overnight at room temperature and then centrifuged at 20 000 × g for 20 min. The crude extract was then collected as a clear supernatant (Nagarsekar et al., 2011).

Determination of total phenolic content

Total phenolics in the A. tortuosum tuber were estimated using the Folin–Ciocalteu’s colorimetric assay. Catechin was used for construction of a standard curve. Total phenolic content was expressed in milligrams of catechin equivalents per gram of the samples (Qingyong & Wai, 1999).

Determination of total flavonoid content

The A. tortuosum tuber extract 100 µl (1 g/mL) was added to equal volumes of 2% AlCl₃ċ6H₂O (2 g in 100 mL methanol) solution. The mixture was shaken vigorously and incubated for 10 min before the absorbance was read at 430 nm. Rutin was used as the standard for the calibration curve, by which a linear equation was derived to determine the total flavonoid content of the samples. Total flavonoid data were expressed in mg of rutin equivalents per gram of dry weight (Bilusic et al., 2005).

Phytochemical analysis

TLC and HPTLC were performed on silica gel 60 f-_254, 20 × 10 cm HPTLC plates (Merck, Germany-#5642) with ethyl acetate--methanol--formic acid--water as 20:2.5:2.5:05 (v/v) as a mobile phase. The A. tortuosum tuber methanol extracts and standards (quercetin, rutin, luteolin and lectin) solutions (5.0 µL of each concentration 1 mg/mL) were applied to the plates as 10 mm bands, sample application with CAMAG-Linomat IV automated spray on band applicator equipped with a 100 µL syringe and operated with the following settings: band length 10 mm, application rate 10 sec/µL, distance between 4 mm, distance from the plate side edge 1.5 cm and distance from the bottom of the plate 2 cm. After development, the plates were air-dried for 15 min. A CAMAG TLC Scanner 3 was used to densitometrically to quantify the bands using WIN CATS software (Version 4 X). The scanner operating parameters were: Mode: absorption/reflection; the slit dimensions; 5 × 0.1 mm; scanning rate: 20 mm/s and monochromatic band width: 20 nm at an optimized wavelength 254, 366 nm, and in the visible range (Huang et al., 2008; Yu et al., 2011).

Determination of antioxidant activity

For antioxidants assays, previously described methods like DPPH scavenging, ABTS radical scavenging activity and FRAP (ferric reducing antioxidant power) were studied using A. tortuosum tuber methanol extract (1 mg/mL). All measurement was done in triplicate. Trolox was used as standard and the standard curve was linear between 25 and 100 µg/mL of Trolox. The percentage inhibition of free radical after sample treatment was calculated. The IC₅₀ values obtained graphically represent the concentration of sample required to scavenge 50% of free radicals (Zouari et al., 2011).

Determination of anti-inflammatory activity

Diene-conjugate assay

Conjugated diene assay involves the preparation of RBC cell membrane. The blood samples were collected with the addition of EDTA (2 mg/mL) as an anticoagulant, then centrifuged, and the plasma was aspirated. The blood cells (0.5 mL) were washed three times using saline (0.89%). Next, 7 mL of ice-cold distilled water were added and left overnight at 0 °C. The hemolysate was separated by centrifugation at 10 000 rpm for 20 min in the cold. The pellet was washed twice with distilled water followed by centrifugation for 10 min and then suspended to a volume of 10 mL of Tris-HCl buffer (0.1 M, pH 7.4) and the resultant solution was then used as a membrane solution. The membrane solution (1.0 mL) was mixed with 5 mL of chloroform: methanol (2:1) and 25 to 100 mg of tuber extract. Each mixture with different concentrations (25 to 100 mg) was centrifuged at 10 000 rpm for 15 min to yield two phases. The chloroform layer was removed using a separating funnel and dried at 45 °C in a water bath. The lipid residue was dissolved in 1.5 mL of cyclohexane and hydroperoxides generated were detected at 233 nm spectrophotometrically against cyclohexane as blank. Salicylic acid (1 mM) was used as a standard drug. The percent activity in all the parameters was calculated by using the standard formula: % Activity = (1-T/C) × 100, where, T and C = absorbance of test and control samples, respectively (Barreca et al., 2011; Stratil et al., 2006).

β-Glucuronidase inhibition assay

For this assay, 2.5 mM p-nitrophenyl- β-d-glucopyranosiduronic acid was incubated with 1 mL (25 to 100 mg) of tuber extract in acetate buffer (0.1 M, pH 7.4) for 5 min followed by the addition of 0.1 mL of β-glucuronidase solution. Each mixture of tuber extract with different concentrations (25 to 100 mg) was further incubated for 30 min followed by addition of 2 mL NaOH (0.5 N) for termination of the reaction. The amount of reaction product formed was observed and absorbance was recorded spectrophotometrically at 410 nm. Salicylic acid (1 mM) was used as a reference drug (Gacche & Dhole, 2006).

Determination of antiproliferative activity

HeLa cells were grown in 75 cm² flasks in MEM with 2 mM l-glutamine, 17.8 mM NaHCO₃, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate and 10% FBS. Cells were seeded into 96-well plates at a density of 5 × 10⁻³ per well, allowed to attach overnight in 300 µL medium and incubated at 37 °C, 5% CO₂. The tuber extract (25 to 100 mg) in 21.7 mM methanol was sonicated for 30 min and sterile-filtered prior to addition to plated cells. Arisaema tortuosum tuber extracts (25 to 100 mg) were added at a final concentration of 0.5, 1, 2 or 5 mg/mL of medium, and the cells left to incubate for 72 h at 37 °C and 5% CO₂. A set of solvent controls (21.7 mM ethanol) were included in each microtitre plate. After incubation, traces of tuber extract were removed by washing the cells twice with 200 µL PBS and applying 100 µL of fresh medium plus 10 µL of 12 mM MTT dissolved in PBS to determine the effects of the tuber extracts on cell proliferation. Cells were then incubated for 4 h at 37 °C, 5% CO₂. To solubilize the product of MTT cleavage, 100 µL of isopropanol containing 0.04 N HCl was added to each well and thoroughly mixed using a multichannel pipette. Within 1 h of HCl-isopropanol addition, the absorbance at 570 nm, with a reference wavelength of 630 nm, was read using a Multiskan Ascent microplate reader (Thermo Labsystems, Franklin, MA). The percent inhibition of cell proliferation was calculated as: Each concentration of the tuber extract was assayed in triplicates (Parry et al., 2006; Wu et al., 2006).

Statistical analysis

All data were expressed as means ± SEM for three replicates for studied parameters. One-way analysis of variance (ANOVA) was used to test for differences between different concentrations.

Results and discussion

Total phenolic and flavonoid content

Phenolics and flavonoids are groups of secondary metabolites which are synthesized by plant tubers; they have been indicated to have several biological activities such as antioxidant, antiaging, antidiabetic, antimutagenic, anticarcinogenic, anti-inflammatory and antimicrobial (Nile & Khobragade, 2009). The amount of total phenolics, measured by Folin–Ciocalteu method and the total phenolic contents obtained from the calibration curve [Y = 84.123X − 5.2640, R²= 0.9992, X is the absorbance; Y is the concentration of catechin solution (l g/mL) and expressed as catechin equivalents (mg catechin/g dried extract], were 86.2 (mg/100 g). The total flavonoid content (mg/mL) was obtained using the regression calibration curve (Y = 0.0005X − 0.0109, R²= 0.995, X is the absorbance; Y is the concentration of rutin solution, l g/mL) and expressed as rutin equivalents (mg rutin/g dried extract). Total flavonoids were found to be 175.5 (mg/100 g) in A. tortuosum tuber extract. Phenols and flavonoids are common groups of polyphenolic compounds and have important roles in stabilizing lipid peroxidation due to their antioxidative activities. Many studies have indicated that the antioxidant capacities of flavonoids are due to the number and position of hydroxyl groups in their structures (Biswas et al., 2010; Heim et al., 2002).

TLC and HPTLC profile

TLC and HPTLC chromatograms for A. tortuosum tuber were developed using a mobile phase (ethyl acetate:acetic acid:formic acid:water) and the plates were subjected to spraying with natural product reagent (NP/PEG). Figure 1 indicates the TLC profile, and Figures 2 and 3 present the HPTLC profile for A tortuosum tuber, respectively. The typical intense fluorescent colored bands were observed on silica gel plates immediately after spraying this NP-PEG at UV-365 nm and when all plates were observed at UV-254 nm fluorescence quenching was observed, which is seen as dark blue zones on the yellow background of the TLC plates. The florescence bands of most of the phytochemicals are not visible at 254 nm but they are visible at 366 nm (Yuan et al., 2005). Depending on the structural type of the phytochemicals different color bands like yellow, blue and green colors were observed. For HPTLC analysis the four different mobile phases previously described for the separation of phytochemicals were tested, using silica gel HPTLC plates, namely, ethyl acetate:formic acid:water (6:1:1, v/v; ethyl acetate:formic acid:acetic acid:water (100:11:11:26), v/v, ethyl acetate:methyl ethyl ketone:formic acid:water (50:30:10:10), v/v, and ethyl acetate:formic acid:water (82:9:9, v/v). The only phase that allowed us to visualize differences among the extracts studied was the mobile phase ethyl acetate: methanol:formic acid:water (20:2.5:2.5:05 v/v). The use of these solvent systems provides good separation of the phytochemicals with respective to solvent system (Figures 2 and 3) (Amin et al., 2006; Lai et al., 2009). This mobile phase offers an improvement over the earlier described methods and may provide the basis for separation of flavonoids, lectins and saponins (Zou et al., 2004). Quercetin, lectin and rutin, (R_f 0.97, 0.62 and 0.53) were found in A. tortuosum tuber (Figures 2 and 3).

Figure 1. TLC profile of the methanol extract of A. tortuosum tuber.

Figure 2. HPTLC profile of the methanol extract of A. tortuosum tuber at 254 nm.

Figure 3. HPTLC profile of the methanol extract of A. tortuosum tuber at 366 nm.

Antioxidant activity

There are many different methods for determining antioxidant function, each of which depends on a particular generator of free radicals, acting by different mechanisms (Wagner & Bladt, 2001). In this study, the A. tortuosum tuber extract showed DPPH scavenging activity (Table 1) and the IC₅₀ value was 852 μg/mL. This IC₅₀ value is comparable with other findings described in the literature for tubers, such as Lycopus lucidus and Thymus algeriensis, which showed scavenging activity against DPPH radicals with IC₅₀ values of 950 and 800 μg/mL, respectively (Males et al., 2004; Yen & Chen, 1995). The antioxidant effect of plant tubers cannot be attributed to their major constituents because minor compounds are likely to play a significant role in the observed activity, and synergistic effects have also been reported (Re et al., 1999). The ABTS coloring method is another commonly used assay to evaluate the antioxidant activity of different substrates in vitro. Reduction of ABTS^-+ radical cations can be even more efficient than that of DPPH (Benzie & Strain, 1996). The ABTS radical scavenging activity of A. tortuosum tuber extract is depicted in Table 1, and this result demonstrated that the tuber extract has a good ability to scavenge ABTS radicals, displaying an IC₅₀ value of 532 μg/mL. Comparing the IC₅₀ values of the ABTS (532 μg/mL) with those of DPPH (852 μg/mL) assays, suggesting that the mechanism of its antioxidant activity is principally based on single electron transfer. Many reports have been demonstrated that the reducing power of natural plant extracts might be strongly correlated with their antioxidant activities (Nile & Khobragade, 2009). Thus, based on this evidence, the FRAP assay was also used to determine the reducing power of A. tortuosum tuber extract to elucidate the relationship between its antioxidant effect and its reducing power. As shown in (Table 1), the tuber extract exhibited ferric-reducing ability (458 μg/mL) and the reducing power was improved by increasing its concentration. These results are in agreement with the findings from the ABTS assay and confirm that the antioxidant power of the tuber extract can be due to an electron transfer mechanism exerted on ABTS radical. The molecular and biochemical mechanisms of the cytotoxicity of tuber cannot be fully understood from the results of this study, but for the reasons given above, it is likely that it acts by inhibiting human colon cancer cell survival.

Table 1. Antioxidant activity of A. tortuosum tuber extract.

	IC50 (μg/mL)
Samples	DPPH	ABTS	FRAP
Tuber extract	852 ± 1.3	532 ± 2.1	458 ± 1.5
Trolox	503 ± 1.6	404 ± 3.1	315 ± 1.3

Values represent mean ± S.E from three experiments.

Anti-inflammatory activity

Anti-inflammatory activity (Table 2) was determined by using diene-conjugate and β-glucuronidase assays. The A. tortuosum tuber extracts revealed an anti-inflammatory activity profile at concentrations of 25, 50, 75 and 100 mg/mL. The inhibition is dose-dependent showing inhibition in the range of 48.2 to 86.20% for diene-conjugate and 52.8 to 78.84% for β-glucuronidase assay, respectively. The inhibition percentages for the control by dieneconjugate and β-glucuronidase assays were 58.00 and 78.84% for salicylic acid. The enzyme β-glucuronidase is present in the lysosomes of neutrophils and has been implicated as one of the mediators in initiating the process of inflammation (Nile & Khobragade, 2009). It is indeed difficult to discuss the exact effect and activity of extract with β-glucuronidase, but one common observation is extracts show high to moderate affinity as compared to standard salicylic acid, which indicates the favoring β-glucuronidase inhibition. The results presented for A. tortuosum tuber revealed excellent anti-inflammatory activities as compared to salicylic acid, a known anti-inflammatory agent. It is known that during inflammation and associated processes, there is an increased production of superoxide ions. It may be possible that the inhibition of superoxide generation in peritoneal macrophages is related to the anti-inflammatory activity of A. tortuosum tuber (Gacche & Dhole, 2006). β-Glucuronidase, which mainly occurs in lysosomes of neutrophils, plays an important role as a mediator in the initiation and progression of inflammation (Nia et al., 2003). Hydroperoxide (dieneconjugates) generation is one of the intermediate steps in membrane lipid peroxidation (Nagao et al., 2001). The lipid peroxidation phenomenon plays a vital role in many inflammatory disorders. Lipid peroxidation results in oxidative modifications of the apoprotein, which is mainly involved in macrophage uptake and atherogenesis (Yuan & Walsh, 2006). Lipid peroxidation is a pro-inflammatory primary event produced by oxidative stress or as a consequence of tissue damage, which can exacerbate tissue injury, due to the potential cytotoxicity and genotoxicity of the end products of lipid peroxidation. Membrane lipids with double bonds are most susceptible to oxidation. Lipid peroxidation can reduce membrane fluidity, leading to increased rigidity throughout the hydrophobic space of membranes, decreased permeability, osmotic fragility and altered activity of certain membrane bound enzymes and transport systems (Barreca et al., 2011; Gacche & Dhole, 2006). Conjugated diene (hydroperoxides) formation is one of the intermediate steps during lipid peroxidation that takes place as a result of the hydrogen capture from the unsaturated fatty acids (Gacche & Dhole, 2006; Stratil et al., 2006). The result indicates that the Arisaema tortuosum tuber may reduce lipid peroxidation by virtue of its antioxidant and anti-inflammatory activity.

Table 2. Anti-inflammatory activities of A. tortuosum tuber extracts.

	Inhibition (%)
Tuber extracts	Diene-conjugate assay	β-Glucuronidase assay
25 mg/mL	48.2 ± 0.51	52.8 ± 0.28
50 mg/mL	62.1 ± 0.42	71.00 ± 0.56
75 mg/mL	78.20 ± 0.53	84.20 ± 0.81
100 mg/mL	86.20 ± 0.32	92.92 ± 0.62
Salicylic acid	58.00 ± 0.72	78.84 ± 0.48

Values represent mean ± S.E from three experiments.

Antiproliferative activity

The A. tortuosum tuber inhibited HeLa cell proliferation in a dose-dependent manner during the 72 h incubation period (Figure 4). For the A. tortuosum tuber extract, after 72 h incubation, inhibition of HeLa cell proliferation was greatest (p < 0.001) with the A. tortuosum tuber extract 100 mg/mL treatments and least with the 25 mg/mL dose. Correlation coefficient analyses showed a positive relationship between A. tortuosum tuber extract polyphenol content and inhibition of HeLa cell proliferation. The trend was strongest at the 100 mg/mL concentration of A. tortuosum tuber extracts: r = 0.965, p = 0.026, but weaker at 25 mg/mL (r = 0.825, p = 0.068). There were no significant relationships between A. tortuosum tuber extract reducing activity and inhibition of HeLa cell proliferation. Deregulation of cell proliferation, together with suppressed apoptosis, is a minimal, common platform for all cancer evolution and progression. Uncontrolled cell division is the primary key in the progression of cancer tumors (Evan & Vousden, 2001). In order to evaluate A. tortuosum tuber as a potential therapy for cancer.

Figure 4. Inhibition of cell proliferation of HeLa cells after 72 h incubation by A. tortuosum tuber extract. Values are significant at (p < 0.026–0.068) between concentrations of extract.

Conclusion

In conclusion, the results indicate that the methanol extracts of A. tortuosum tubers possessed high antioxidant, anti-inflammatory, and antiproliferative effects in vitro and can be an easy accessible source of natural antioxidant, anti-inflammatory, and antiproliferative agent. However, the components responsible for antioxidant activity of tuber extracts are unclear. Further bioguided fractionation of the active phytochemicals from A. tortuosum tubers is being carried out, aiming for the formulation of a safer and efficient drug to prevent the oxidative stress and related diseases. The A. tortuosum tubers extract revealed significant anti-inflammatory and antiproliferative activity, which indicates that the phytochemicals present in A. tortuosum tubers extract may be useful for the treatment of diseases like gout, hyperuricemia, and cancer, respectively, which are correlates with the ethno- botanical data on the use of this plant in Indian folklore and ayurveda. The data provides a basis for further investigation to isolate the active constituents from A. tortuosum tuber and drug development against diseases related to oxidative stress and inflammation.

Declaration of interest

This research was supported by the 2012 KU-Brain Pool Program of Konkuk University, Seoul, South Korea. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.