|Year : 2019 | Volume
| Issue : 1 | Page : 38-47
Studies on phytoconstituents, in vitro antioxidant, antibacterial, antiparasitic, antimicrobial, and anticancer potential of medicinal plant Lasiosiphon eriocephalus decne (Family: Thymelaeaceae)
Pratik P Durgawale, Madhavi N Patil, Shreepad A Joshi, Kalpita S Korabu, Kailas D Datkhile
Department of Molecular Biology and Genetics, Molecular and Genetic Laboratory, Krishna Institute of Medical Sciences University, Satara, Maharashtra, India
|Date of Web Publication||4-Feb-2019|
Kailas D Datkhile
Department of Molecular Biology and Genetics, Molecular and Genetic Laboratory, Krishna Institute of Medical Sciences (Deemed To Be University), Taluka-Karad, Satara - 415 110, Maharashtra
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: The present study has been undertaken with an objective to determine the antioxidant, antimicrobial, antiparasitic, and cytotoxic activity of endangered medicinal plant Lasiosiphon eriocephalus. Materials and Methods: Aqueous, methanol, and ethanol extracts of leaves, stem bark, and flowers of the selected medicinal plant were screened for the presence of phenolics, tannins, and flavonoids. In vitro antioxidant activity was evaluated by 1,1-diphenyl-2-picryl hydrazine and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) assay. The antiparasitic activity of extracts was tested against protozoan endoparasite Leishmania donovani, and antimicrobial activity plant extracts were determined against Klebsiella pneumonia, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The anticancer properties of plant extracts were studied by cytotoxicity on cancer cells including HeLa and MCF-7 determined by MTT assay and DNA fragmentation assay. Results: The extracts screened for phytochemical analysis was found to contain phenolics, tannins, and flavonoids. The water and solvent extracts of leaves, and flowers had shown strong antioxidant activity in vitro. The leaves and flower extract showed the ability to inhibit the growth of both Gram-positive as well as Gram-negative bacteria. The results also showed increased antimicrobial and antiparasitic activities of ethanol extracts of flowers then leaves and followed by bark. The present study revealed that the treatment of crude extract of leaves as well as stem bark of L. eriocephalus exhibited strong cytotoxic activity against tested cancer cell lines. Conclusion: These results indicated that the possible secondary metabolites from the crude extract of L. eriocephalous plant have a potential as antioxidant, antiparasitic, antibacterial, and cytotoxic agent against cancer cells. However, further study needs to be carried out to understand the detailed molecular mechanism involved in the inhibition of cell proliferation and induction of apoptosis in response to phytoconstituents derived from L. eriocephalus.
Keywords: Antimicrobial activity, antioxidants, antiparasitic activity, cytotoxicity, Lasiosiphon eriocephalus
|How to cite this article:|
Durgawale PP, Patil MN, Joshi SA, Korabu KS, Datkhile KD. Studies on phytoconstituents, in vitro antioxidant, antibacterial, antiparasitic, antimicrobial, and anticancer potential of medicinal plant Lasiosiphon eriocephalus decne (Family: Thymelaeaceae). J Nat Sc Biol Med 2019;10:38-47
|How to cite this URL:|
Durgawale PP, Patil MN, Joshi SA, Korabu KS, Datkhile KD. Studies on phytoconstituents, in vitro antioxidant, antibacterial, antiparasitic, antimicrobial, and anticancer potential of medicinal plant Lasiosiphon eriocephalus decne (Family: Thymelaeaceae). J Nat Sc Biol Med [serial online] 2019 [cited 2019 Jun 16];10:38-47. Available from: http://www.jnsbm.org/text.asp?2019/10/1/38/251501
| Introduction|| |
Naturally occurring phytochemicals from medicinal plants contain a wide range of chemical substances with important therapeutic properties which have acquired substantial contribution in the treatment of deadly diseases. According to the World Health Organization, more than 80% of the world's population relies on traditional medicine for their primary health-care needs. The plant kingdom holds many plant species containing substances of medicinal value which have yet to be discovered. Therefore, nowadays, interest has been developed to elucidate the mode of action of number of phytochemicals from different medicinal plants. Several studies have been conducted to reveal their antioxidant,, antibacterial,,, and antiparasitic, properties. In recent years, medicinal plants and plant-derived products have also attracted researchers due to their diverse range of therapeutic properties in the management of various cancers.,,
Lasiosiphon eriocephalus Decne commonly known as Raamethaa (Family: Thymelaeaceae) is widely used in the treatment of many aliments. It has widely distributed all over India, mostly in Maharashtra, Kerala, Karnataka, and throughout Western Ghats. Phytochemical studies on several species of family Thymelaeaceae showed the presence variety of different classes of natural products including secondary metabolites such as tannins, glycosides, coumarins, and flavonoids linearly distributed in plant parts such as leaves, stem bark, roots, and fruits.,,, There are few reports on phytochemicals and their pharmacological activities in different plant parts of these plants. Earlier, several genera of Thymelaeaceae are considered for their biological activities such as anticancer, antileukemic, antidiabetic, antiplasmodial, and cytotoxicity activities. Similarly, phytochemicals from different species of Thymelaeaceae have been reported to show antioxidant activity in Gnidia stenophylla, antibacterial in (Gonia capitata),, antifungal in (G. kraussianus), and antiparasitic in (G. cuneata) activities., Furthermore, L. eriocephalus have tremendous pharmacological potential which had been used traditionally for long time as substitutional medicine for a variety of medicinal complaints in humans including dropsy, sores, burns, constipation, coughs, earache, epilepsy, headache, fevers, insanity, malaria, measles, pulmonary tuberculosis, smallpox, snake bites, sprains and fractures, tonsillitis, stomach and chest complaints, toothache, and ulcers.
Although earlier studies reported curative properties of active phytoconstituents from other genera of family Thymelaeaceae including Daphne, Gnidia, Wikstroemia, and Pimelea species, lesser efforts have been made to elucidate either of antimicrobial, antiparasitic, cytotoxicity, or genotoxicity properties of L. eriocephalus. Therefore, in this study, we selected this traditional endangered medicinal plant with an aim investigate the effect of different solvents extractions on phytochemicals and to evaluate antioxidant, antibacterial as well as antiparasitic properties of different solvent extracts of L. eriocephalus. This study was further extended to study cytotoxic and genotoxic properties of different solvent extracts of leaves, stem bark, and flowers of L. eriocephalus.
| Materials and Methods|| |
Chemicals and reagents
2, 2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 1,1-diphenyl-2-picryl hydrazine (DPPH) were obtained from Sigma Aldrich. Butylated hydroxytoluene (BHT), deoxyribose, nitroblue tetrazolium, Phenazine methosulfate, ascorbic acid, Potassium persulfate, phosphate buffer, potassium ferricyanide, thiobarbituric acid, trichloroacetic acid, ferric chloride, ethylenediaminetetraacetic acid (EDTA), and the solvents were obtained from Sisco Research Laboratories. Minimum Essential Medium (10370-021), fetal bovine serum (FBS) (2614079), RPMI1640 (11875-085), and penicillin-streptomycin (15140-122) were purchased from Thermo Fisher Scientific, DMSO (D2650), purchased from Sigma Aldrich, MTT (M6494) from Invitrogen, 6 well plates (CLS3355), 96 well plates (CLS3599), and 5 ml serological pipettes (CLS4487) purchased from Corning.
Collection and processing of plant materials
Plant parts were collected from Amboli Ghat region of Maharashtra from India and authenticated by the Department of Botany, University of Pune, India. The plant parts were thoroughly washed with double-distilled water (ddH2O) for removal of adhered dust particles, shade dried and then ground into fine powder using mortar and pestle. The powdered samples were stored in an air-tight container in the dark at room temperature for further experiments.
Preparation of solvent extracts
Based on the published literature, 25 g of powdered leaf, stem bark, and flowers were used for solvent extraction. Soxhlet extraction was carried out in different solvents, namely distilled water, methanol, and ethanol. The extracts obtained were evaporated in crucible dishes kept in desiccators till dryness. The dried extracts were dissolved in their respective solvents to get known concentration of the extracts (10 mg/mL) and preserved at 4°C in airtight bottles until further use.
Phytochemical screening of Lasiosiphon eriocephalus plant extracts of different plant parts
Determination of total polyphenolic content
The total soluble polyphenolic content of plant extracts from different plant parts was determined by the Folin–Ciocalteu reagent method. The reaction mixture containing 100 μL of sample and 750 μL of 7.5% sodium carbonate (Na2 CO3) solution, 250 μL of Folin–Ciocalteu reagent and the reaction was incubated at 37°C for 2 h (h) with intermittent shaking, and the absorbance was measured at 765 nm. Gallic acid (100 μg/mL) was used for calibration of the standard curve. The results were expressed as microgram of gallic acid equivalent (mg GAE)/g of dry plant material.
Determination of tannin content
Total tannin content of plant extracts from different plant parts was determined by the Folin–Ciocalteu reagent method. The reaction mixture containing 100 μL of (10–100 μg/mL) plant extract in 750 μL of ddH2O, 50 μL of Folin–Ciocalteu reagent, and 200 μL of 17.5% Na2 CO3 was mixed thoroughly, and the reaction was incubated at 37°C for 30 min with intermittent shaking and the absorbance was measured at 725 nm using tannic acid as standard. Tannic acid (20–100 μg/mL) was used for calibration of standard curve. The results were expressed as mg of tannic equivalent to Gram of dry plant material.
Estimation of total flavonoid content
The total flavonoid content was determined using the Dowd method. 200 μL of different concentrations of plant extracts in water, methanol, and ethanol were mixed with add 100 μL of a 5% (w/v) NaNO2 and mixed vigorously, After 5 min, 200 μL of 10% (w/v) aluminum trichloride was added in each tube and allowed to stand for a further 10 min at room temperature. 500 μL of 1 M NaOH was added finally and the volume of mixture was adjusted to 2.0 mL with ddH2O. The absorbance of reaction was measured at 510 nm against blank samples. The total flavonoid content was determined as microgram of rutin equivalent by using an equation that was obtained from standard graph using standard rutin solution (10–100 μg/mL) prepared in ethanol.
Evaluation of antioxidant properties of Lasiosiphon eriocephalus plant extracts
Antioxidant properties were determined by free radical scavenging assays such as DPPH and ABTS assay.
1,1-diphenyl-2-picryl hydrazine radical scavenging assay
The DPPH scavenging activity of aqueous, methanol, and ethanol extracts of different plant parts was measured according to the method described by RajanRushender et al., 2012 with some modifications. The reaction mixture consisted of a mixture of freshly prepared 1 mL DPPH (0.1 mM) solution in methanol with different concentrations (25, 50, 100, 200, and 400 μg/mL of aqueous, methanol, and ethanol extracts in final volume of 0.1 mL in distilled water or methanol, respectively. The blank sample consisted of 2 mL of methanol, while the control contained 2 mL DPPH methanolic solution only. The absorbance of sample, blank, and control was measured at 517 nm, after 30 min incubation of reaction in the dark at room temperature, using an ultraviolet (UV)-Visible 1800 spectrophotometer (Shimadzu). The percentage of DPPH scavenging activity was calculated using the expression below: Percentage inhibition (%) = (A0–A1)/A0) ×100, where: A0 is the Absorbance of control and A1 Absorbance of test. The results were compared with ascorbic acid and BHT as standard.
2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging assay
ABTS radical scavenging assay was carried out using procedures described by Arnao et al. 2001. ABTS•+ radical cations are produced by reacting ABTS (7 mM) and potassium persulfate (2.4 mM) and incubating the mixture at room temperature in the dark for 16 h. Different concentrations of aqueous as well as methanolic extracts in final volume of 0.1 mL were added and allowed to react with 1 mL of ABTS for 30 min and the absorbance was recorded at 734 nm after incubation. The blank sample consisted of 1 mL of methanol, while the control contained 1 mL ABTS solution prepared in methanol. Ascorbic acid and BHT were used as standard. The percentage of inhibition was calculated from the following equation:
Percentage inhibition (%) = (A0–A1)/A0) ×Evaluation of antimicrobial properties of Lasiosiphon eriocephalus plant extracts
The antimicrobial activity of plant extracts was performed against Gram-positive Bacteria Staphylococcus aureus (ATCC® 29213™) and Gram-negative bacteria, Escherichia coli (ATCC® 25922™), Klebsiella pneumoniae (ATCC® 700603™) and Pseudomonas aeruginosa (ATCC® 2617™). In a sterile, 1.5 mL microcentrifuge tube, the sample solutions at different concentrations were tested on bacterial solutions in 1 mL of Muller Hinton culture medium incubated at 37°C for 24 h. Ampicillin instead of the sample solution, sterile water instead of bacterial solution as positive control and either methanol or ethanol solvents were used as negative controls for each assay. After incubation period, bacterial growth inhibition (GI) was evaluated by spectrophotometer by checking absorbance at 600 nm. The activity of the sample was calculated as percentage of bacterial % GI according to the following equation: % GI = (Negative correction – Sample correction)/Negative correction × 100. Where, Negative correction: Absorbance of negative control–Absorbance of sterility control; Sample correction: Absorbance of the samples–Absorbance of color control.
Evaluation of antiparasitic properties of Lasiosiphon eriocephalus plant extracts
Leishmania donovani parasites used in the assays were transformed from promastigote to amastigote forms transferring 1 mL of promatigotes log phase culture into 5 mL of RPMI1640 supplemented with 10% heat-inactivated FBS and maintained at 25°C until its use in bioassays. Primary screening of different plant extracts was performed by exposure of L. donovani cells at different concentrations of extracts for 48 h. The inhibition of parasites growth in culture was determined by observing under phase contrast microscope for their morphology.
Evaluation of anticancer properties of Lasiosiphon eriocephalus plant extracts
In vitro evaluation of cytotoxicity properties of Lasiosiphon eriocephalus plant extract
In vitro cytotoxicity effects of L. eriocephalus leaves, stem bark and flower extracts the on human cancer cell lines including HeLa and MCF-7 cells were determined by the MTT assay. The cells were maintained in MEM medium supplemented with 10% FBS, Penicillin-Streptomycin at 100 U/mL, 100 μg/mL in a humidified atmosphere of 5% CO2 at 37°C. In a 96 well plate, 10,000 cells were added in 100 μL of MEM medium and incubated at 37°C, 5% CO2. After 24 h incubation, the confluent cells were exposed to respective treatment of extracts at concentrations of 0.025, 0.05, 0.1, 0.2, 0.4, and 0.8 mg/mL in culture medium without FBS and incubated for further 48 h at 37°C and 5% CO2. After 48 h completion of treatment, cells were washed with sterile Hanks' Balanced Salt Solution thereafter 10 μL per well (10 μL/well) of MTT (5 mg/ml) added to the cells and the plates were further incubated 37°C, 5% CO2 atmosphere. After 4 h incubation, the MTT solution was carefully removed and 200 μL of DMSO was added to each well. The absorbance of purple color developed was measured at 570 nm wavelength using UV-Vis 1800 spectrophotometer (Shimadzu). The results were expressed as percentage of cell survival as compared to the control. All experiments were performed in triplicates. The morphology of the cells was then examined in an inverted phase-contrast microscope (Primovert Carl Zeiss microscope).
DNA fragmentation assay
The apoptotic activity of the extracts of L. eriocephalus was determined by DNA fragmentation assay. HeLa and MCF-7 cells (1 × 106) in 6 well plates were treated with 50% inhibitory concentration (IC50) and 90% inhibitory concentration (IC90) concentration of extracts along with untreated control cells, cells treated with solvents used for preparation of extracts and incubated at 37°C in 5% CO2, for 48 h. Cells were harvested, followed by lysis in 0.3 mL of cell lysis buffer containing (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.2% Triton X-100, and 0.5% sodium dodecyl sulfate). After treatment with 0.5 mg/mL of Ribonuclease A (RNase-A) at 37°C for 1 h, thereafter, 0.2 mg/mL of Proteinase-K at 55°C for 1 h, DNA was precipitated by addition of equal volume of isopropanol and 1/10th volume of 5 Molar (M) sodium chloride. The DNA pellet was resuspended in Tris-EDTA (TE) buffer and DNA was separated on 1.5% (w/v) low-EEO agarose (GeNei) gel containing 1 μg/mL ethidium bromide and subjected to electrophoresis at 80 volts (V) for 1–2 h in Tris-Acetate-EDTA buffer along with 100 base pair (bp) and 1 kilo bp DNA molecular weight marker. The DNA fragments were visualized by exposing the gels to UV transilluminator followed by photography in gel documentation system (BioRad Laboratories, USA).
All experiments were done in three replicates and then values were expressed as mean ± standard error of three measurements. One-way analysis of variance and Student's t-test were carried out using Microsoft Excel 2010 to evaluate the possible differences among the means. There were significant differences with P ≤ 0.05.
| Results|| |
Preliminary phytochemical screening of the aqueous, methanol and ethanol extracts of different plant parts including leaves, stem bark, and flowers of L. eriocephalus revealed the presence of various groups of compounds (phenolics, tannins, and flavonoids). These data were obtained according to the colorimetric analyses quantitated using standard curve of gallic acid, tannic acid, and rutin as standard solutions. The results are summarized in [Figure 1].
|Figure 1: Representative histogram showing (a) total phenolic content (b) total tannin content and (c) flavonoid content in the aqueous, methanol, and ethanol extracts of leaves, stem bark, and flowers of Lasiosiphon eriocephalus. Values are expressed as the means of three independent experiments|
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In vitro antioxidant activity
The in vitro DPPH and ABTS radical scavenging activity of different solvent extracts of L. eriocephalus is shown in [Figure 2]a and [Figure 2]b. The highest DPPH radical scavenging activity was observed in the methanol and ethanol extracts of flowers, while aqueous extract also showed comparatively lower effect. The results showed that the aqueous extract of leaves exhibited comparatively lower DPPH radical scavenging ability (IC50:184.07 ± 23.77 μg/mL) than the methanol (IC50:131.35 ± 15.68 μg/mL) and ethanol extracts (IC50:46.95 ± 3.28 μg/mL). IC50 value of aqueous extract of stem bark and flowers were observed as (118.11 ± 11.93 μg/mL) and (52.22 ± 7.02 μg/mL) and methanol extract (77.34 ± 10.97 μg/mL; 51.81 ± 5.73 μg/mL and ethanol extracts (59.70 ± 0.54 μg/mL) and (44.57 ± 0.51 μg/mL), respectively. Thus, DPPH radical scavenging activity of ethanol extracts of flowers was significantly higher than aqueous and methanol extracts of both stem bark and leaves. Similarly, when we checked ABTS radical scavenging activity of extracts of L. eriocephalus plant parts, aqueous, methanol, and ethanol extracts of flowers showed significantly increased radical scavenging activity than stem bark and leaves. IC50 values of aqueous extract of leaves, stem bark, and flowers were 62.24 ± 1.10 μg/mL; 61.17 ± 0.53 μg/mL; and 42.78 ± 0.77 μg/mL, methanol extract was 25.36 ± 0.42 μg/mL; 30.88 ± 1.55 μg/mL; 23.38 ± 0.74 μg/mL, and ethanol extracts were 21.90 ± 0.72 μg/mL; 19.95 ± 1.05 μg/mL; and 15.06 ± 0.25 μg/mL, respectively.
|Figure 2: Representative histogram showing (a) 1,1-diphenyl-2-picryl hydrazine and (b) 2, 2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging activity of different concentrations of aqueous, methanol, and ethanol extracts of leaves, stem bark, and flowers of Lasiosiphon eriocephalus. The data represent the percentage inhibition of radicals in vitro. The results represent the means of three independent experiments, and error bars represent the standard error of the mean|
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Antimicrobial activity of tested extracts of Lasiosiphon eriocephalus
The antibacterial activities of the aqueous, methanol, and ethanol extracts were assayed in vitro by minimum inhibitory concentration method against four bacteria species, S. aureus, K. pneumoniae, E. coli, and P. aeruginosa. Among the tested solvent extracts, ethanol extract of flowers showed minimum antimicrobial activity in selected bacterial species except K. pneumoniae where aqueous extract of leaves showed 18.99% ± 0.21% of inhibition of bacterial cell growth, whereas methanol and ethanol extracts showed 37.54% ±6.63% and 69.78% ±8.96% inhibition of cell growth at concentration of 1 mg/mL [Figure 3]c. Whereas, aqueous, methanol, and ethanol extracts of stem bark of L. eriocephalus showed 40.66 ± 4.71, 44.36 ± 7.14, and 71.34 ± 0.35 and flowers showed 39.40 ± 0.39, 57.67 ± 5.49, and 75.57 ± 3.70 inhibition K. pneaumoniae cell growth, respectively. The aqueous, methanol, and ethanol extracts of leaves, stem bark, and flowers showed the highest inhibition of E. coli bacterial cell growth at 1.0 mg/mL concentration [Figure 3]a. Similarly, doses of both aqueous, methanol, and ethanol extracts of leaves stem bark and flowers required to kill S. aureus, and P. aeruginosa are lower which showed more than 90% of GI at 1 mg/mL concentration of both leaves, stem bark, and flower extracts[Figure 3]b and [Figure 3]d.
|Figure 3: Representative histogram showing bacterial growth inhibition of (a) Escherichia coli ATCC®25922™ (b) Pseudomonas aeruginosa ATCC® 2617™ (c) Klebsiella pneumoniae ATCC® 70063™ and (d) Staphylococcus aureus ATCC® 29213™ exposed to different concentrations (0.2, 0.5, and 1.0 mg/mL) of aqueous, methanol, and ethanol extracts of leaves, stem bark and flowers of Lasiosiphon eriocephalus for 24 h. The results represent the means of three independent experiments, and error bars represent the standard error of the mean|
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Antiparasitic activity of tested extracts of Lasiosiphon eriocephalus
In the antiparasitic assays, different solvent extracts including water, methanol, and ethanol extracts of leaves [Figure 4]a, stem bark [Figure 4]b, and flower [Figure 4]c were tested against L. donovani. Among all the extracts tested, methanol and ethanol extract of bark and flowers showed the highest antiparasitic activity at the dose of 0.6 mg/mL and 0.4 mg/mL, respectively.
|Figure 4: Cell morphology of Leishmania donovani cells after 24 h treatment of aqueous, methanol and ethanol extracts of (a) leaves, (b) stem bark, and (c) flowers of Lasiosiphon eriocephalus at different concentrations. All images are taken at ×20 magnification with Carl Zeiss phase contrast microscope. Scale bars, 100 μm|
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Anticancer activity of tested extracts of Lasiosiphon eriocephalus
In order to screen cytotoxicity of aqueous as well as ethanol and methanol extracts of leaves, stem bark and flowers of L. eriocephalus, the cell viability was determined by MTT assay. The cytotoxic activity of L, eriocephalus extracts on MCF7 cells represented in figure [Figure 5]a and [Figure 5]b whereas the activity of extracts on HeLa cells shown in [Figure 5]c and [Figure 5]d. When plant extract was tested for cytotoxic activity, the data obtained from cell viability results of HeLa and MCF-7 cells revealed that methanol extract of leaves and stem bark exhibited a range of significant cytotoxic activities in a dose-dependent manner varying from 0.025–0.6 mg/mL whereas aqueous extract of leaves and stem bark showed decreased cell viability with an increase in the concentration of the extract from 0.025–0.8 mg/mL. The results indicated that when MCF-7 cells were treated with 0.4 mg/mL concentration of methanol and ethanol extract of leaves, stem bark and flowers, the cells showed 100% mortality whereas 0.8 mg/mL concentration of aqueous extract was needed to kill MCF-7 cells. HeLa cells required 0.6 mg/ml of methanol as well as ethanol extract for 100% inhibition. Similarly, 0.8 mg/ml of aqueous extracts of leaves, stem bark and flowers was required to kill MCF-7 and HeLa cells. Microscopic observations of aqueous, methanol as well as ethanol extract of leaves, stem bark, and flowers treated MCF-7 and HeLa cells showed distinct cellular morphological alterations such as loss of membrane integrity therefore appeared shrunken, cytoplasmic condensation as indicating unhealthy cells, whereas the control cells appeared normal [[Figure 5]b and [Figure 5]d, respectively]. The evaluation of genotoxic effects of IC50 and IC90 concentrations of methanol as well as aqueous extracts of both leaves and stem bark of L. eriocephalus on cancer cells including HeLa and MCF-7 cells were assessed by DNA fragmentation pattern on agarose gel. The methanol and aqueous extract of leaves and stem bark was significantly genotoxic to MCF-7 and HeLa cells as noted by extensive double-strand breaks thereby yielding a ladder appearance when treated with IC50 and IC90 as concentration of extracts as shown in [Lanes 3 and 4, [Figure 6]] while the DNA of control cells exhibited minimum or no DNA breakage [Lane 2, [Figure 6]] of MCF-7 and HeLa cells.
|Figure 5: (a) Representative histogram showing in vitro dose-dependent cytotoxicity of (A) aqueous extract of leaves, bark, and flowers on MCF-7 cells exposed for 24 h, (B) MCF-7 cells exposed to methanol extracts of plant parts exposed for 24 h, (C) MCF-7 cells exposed to ethanol extracts for 24 h, (D) MCF-7 cells exposed to aqueous extracts of plant parts exposed for 48 h, (E) MCF-7 cells exposed to methanol extracts of plant parts exposed for 48 h (F) MCF-7 cells exposed to ethanol extracts of plant parts exposed for 48 h. The results represent the means of three independent experiments, and error bars represent the standard error of the mean. (b) Cell morphology of MCF-7 cells after 48 h treatment of aqueous, methanol, and ethanol extracts of leaves, stem bark, and flowers of Lasiosiphon eriocephalus at different concentrations. All images are taken at ×20 magnification with Carl Zeiss phase contrast microscope. Scale bars, 100 μm. (c) Representative histogram showing in vitro dose dependent cytotoxicity of (A) aqueous extract of leaves, bark and flowers on HeLa cells exposed for 24 h, (B) HeLa cells exposed to methanol extracts of plant parts exposed for 24 h, (C) HeLa cells exposed to ethanol extracts for 24 h, (D) HeLa cells exposed to aqueous extracts of plant parts exposed for 48 h, (E) HeLa cells exposed to methanol extracts of plant parts exposed for 48 h (F) HeLa cells exposed to ethanol extracts of plant parts exposed for 48 h. The results represent the means of three independent experiments, and error bars represent the standard error of the mean. (d) Cell morphology of HeLacells after 48 h treatment of aqueous, methanol, and ethanol extracts of leaves, stem bark, and flowers of Lasiosiphon eriocephalus at different concentrations. All images are taken at ×20 magnification with Carl Zeiss phase contrast microscope. Scale bars, 100 μm|
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|Figure 6: Representative agarose gel images showing DNA fragmentation in MCF-7 cells (Left panel) and HeLa cells (Right panel) treated with aqueous, methanol, and ethanol extracts of leaves, bark, and flowers of Lasiosiphon eriocephalus at IC50and IC90concentrations. In each representative gel, lane 1 is 100 bp DNA marker: Lane 2 is DNA from control cells followed by lane 3: DNA from IC50concentration treated cells, lane 4: DNA from IC90treated cells and lane 5: Is 1 kilo base pair DNA marker|
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| Discussion|| |
In the present study, phytochemical screening, in vitro antioxidant, antimicrobial, and cytotoxic activities of different solvent extracts from the L. eriocephalus were examined. Phytochemical evaluation was performed with crude aqueous, methanol and ethanol extracts of leaves, stem bark and flowers of L. eriocephalus where both methanol and ethanol extracts showed the presence of higher amounts of phytochemicals including phenolics, tannins, and flavonoids. Both methanol and ethanol extracts of flowers were found to be rich in phenols and tannins than aqueous extract. The tested extracts have a strong antioxidant activity against various oxidative systems in vitro including DPPH and ABTS radicals. We found that the tested extracts exhibited the higher radical scavenging activity with the greatest amount of phytoconstituents content. Extensive research has been carried out on the free radical scavenging properties of phytochemicals of different parts of various medicinal plants., However, the components responsible for the antioxidative activity of L. eriocephalus extracts are currently unclear. Earlier, only a single report available on antioxidant properties of Gnidia glauca from the family Thymelaeaceae stating the antioxidant capacity in stem bark and leaves, but in contrary to that report our results showed marginal increase in antioxidant capacity in the flowers. The phytochemical analysis of L. eriocephalus already revealed the presence of secondary metabolites including phenolic, tannins, and flavonoid compounds in this plant extracts could be contributed to its antioxidant properties. Thus, the in vitro antioxidant activity of L. eriocephalus indicates that the plant extracts may contain compounds that are capable of donating hydrogen to a free radical to remove electrons responsible for the radical's reactivity.
Antimicrobial properties of medicinal plants are being increasingly reported from different parts of the world., In the present study, among all the tested samples of L. eriocephalus, both ethanol and methanol extract of leaves, bark, and flowers showed bacterial growth inhibitory effects against bacterial strains except K. pneumoniae. Earlier, roots, seeds, or bark extracts of L. eriocephalus showed antibacterial activity, but the higher concentrations of plant extracts are needed to inhibit bacterial cell growth. In contrast to those, our results showed that the lower concentrations of leaf and flowers showed more bacterial GI against E. coli and P. aeruginosa. The antimicrobial assay revealed that ethanol extract of flowers and leaves of L. eriocephalus exhibited broad-spectrum activity against tested isolates as compared to aqueous extracts of selected plant parts. The results obtained from this study indicated that the plant extracts showed the stronger antimicrobial activity than the commercially available antibiotics. When we compared antiparasitic activity of different parts of L. eriocephalus, earlier evaluations of few genera of Thymelaeaceae including Gnidia krasssianus and G. cuneata, showed the absence of antiparasitic activities. In contrast to those findings, our results showed strong antiparasitic properties activity of different solvent extracts of L. eriocephalus against L. donovani.
In this study, cytotoxicity of the plant parts of L. eriocephalus leaves, stem bark, and flowers was also investigated on MCF-7 and HeLa cells. Analyses of the cytotoxicity of the ethanol extract of flowers and leaves demonstrated considerable activity as exhibited by an IC50 value of 0.033 mg/mL and 0.061 mg/mL, followed by the activity of ethanol extract of stem bark with IC50 0.084 mg/mL. The methanol extract of flowers showed strong cytotoxic activity with IC50 0.05 mg/mL whereas the cytotoxic effects of methanol extracts of leaves and stem bark showed comparatively lower cytotoxic effects on MCF-7 cells with IC50 valves of 0.150 mg/mL and 0.108 mg/mL, respectively. Comparatively, the doses of aqueous extracts of leaves, stem bark, and flowers required to kill both HeLa and MCF-7 cells are higher with IC50 Leaves: 0.523 mg/mL, Stem bark: 0.327 mg/mL, and Flowers: 0.128 mg/mL for HeLa cells and IC50 Leaves: 0.313 mg/mL, Stem bark: 0.210 mg/mL, and Flowers: 0.144 mg/mL for MCF-7 cells. The crude ethanol extract of the leaves gave the highest efficacy toward HeLa cells with a half maximal inhibitory concentration of 0.024 mg/mL, which was followed by the flowers and stem bark extracts which gave IC50 values of 0.034 and 0.083 mg/mL, respectively. The methanol extract of flowers and leaves were moderately cytotoxic and was able to decrease cell viability by 50% at 0.047 mg/mL and 0.066 mg/mL, respectively, whereas methanol extract of stem bark showed comparatively lower doses for cytotoxicity for HeLa cells. The results clearly indicated that both methanol, as well as ethanol extracts of leaves, stem bark, and flowers, showed significant inhibitory effects on the tested HeLa as well as MCF-7 cells at comparatively lower concentrations than the aqueous extracts. A number of studies have noted cytotoxic activities of traditional medicinal plants which had not been reported before.,, In our experiments, the MCF-7 and HeLa cells when treated with both aqueous extracts, methanol, and ethanol extracts for 48 h, the results showed dose-dependent cytotoxicity of leaves, stem bark, and flower extracts against tested cells. We further analyzed the DNA fragmentation, considered as one of the hallmarks of cell apoptosis was confirmed resolving DNA extracted from MCF-7 and HeLa cells exposed to IC50 and IC90 concentrations of aqueous, methanol as well as ethanol extracts of leaves, stem bark, and flowers on agarose gels. Extensive DNA double-strand breaks appeared from MCF-7 and HeLa cells exposed to plant extracts which serve as the powerful genotoxic potential of L. eriocephalus. Thus, our study provides plausible evidence supporting L. eriocephalus plant extract exhibited strong cytotoxic and apoptotic potential against selected cancer cells. Further detailed studies are necessary to enhance the molecular mechanism responsible for cytotoxicity, antiproliferative, and apoptotic effects of genotoxic compounds in this plant.
| Conclusion|| |
In the present study, ethanol extract of flowers and leaves of L. eriocephalus showed maximum antioxidant, antibacterial, antiparasitic, and cytotoxic activity. The phytochemical analysis shows the presence of flavonoids, tannins, and phenols with high antioxidant activity. Antibacterial activity of the flowers, as well as leaves and stem bark, shows the ability to inhibit the growth of Gram-positive as well as Gram-negative bacteria. The cytotoxicity of different solvent extracts on cancer cell lines showed strong cytotoxic and genotoxic effects. The obtained results could form a good basis for further investigation in the potential discovery of new natural bioactive compounds and molecular mechanism involved in cytotoxicity and genotoxicity of those bioactive compounds from this traditional plant with medicinal value.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Bharti R, Ahuja G, Ganapathy PS, Dakappa SS. A review on medicinal plants having antioxidant potential. J Pharm Res 2012;5:4278-87.
Nigam V, Sodhi JS. Some medicinal plants with antioxidant activity-A review. Int J Pharm Biol Sci 2014;4:173-8.
Vashist H, Jindal A. Antimicrobial activities of medicinal plants – Review. Int J Res Pharm Biomed Sci 2012;3:222-30.
Munuswamy H, Thirunavukkarasu T, Rajamani S, Kuppan E, Ernest DA. Review on antimicrobial efficacy of some traditional medicinal plants in Tamilnadu. J Acute Dis 2013;2:99-105.
Compean KL, Ynalvez RA. Antimicrobial activity of plant secondary metabolites: A review. Res J Med Plant 2014;8:204-13.
Bauri RK, Tigga MN, Kullu SS. A review on use of medicinal plants to control parasites. Ind J Nat Prod Res 2015;6:268-77.
Al-Snafi AE. Antiparasitic effects of medicinal plants (part 1) - A review. IOSR J Pharm 2016;6:51-66.
Wang H, Khor TO, Shu L, Su ZY, Fuentes F, Lee JH, et al.
Plants vs. cancer: A review on natural phytochemicals in preventing and treating cancers and their druggability. Anticancer Agents Med Chem 2012;12:1281-305.
Prakash O, Kumar A, Ajeet KP. Anticancer potential of plants and natural products: A review. Am J Pharmacol Sci 2013;1:104-15.
Shukla S, Mehta A. Anticancer potential of medicinal plants and their phytochemicals: A review. Braz J Bot 2015:38:199-210.
Cottiglia F, Loy G, Garau D, Floris C, Casu M, Pompei R, et al.
Antimicrobial evaluation of coumarins and flavonoids from the stems of Daphne gnidium
L. Phytomedicine 2001;8:302-5.
Li SH, Wu LJ, Gao HY, Chen YH, Li Y. A new dicoumarinoid glycoside from Daphne giraldii
. J Asian Nat Prod Res 2005;7:839-42.
Pan L, Zhang XF, Wu HF, Ding LS. A new daphnane diterpene from Daphne tangutica
. Chin Chem Lett 2006;17:38-40.
Liang S, Tang J, Shen YH, Jin HZ, Tian JM, Wu ZJ, et al.
Biflavonoids from Daphne feddei
and their inhibitory activities against nitric oxide production. Chem Pharm Bull (Tokyo) 2008;56:1729-31.
Borris RP, Blasko G, Cordell GA. Ethnopharmacologic and phytochemical studies of Thymelaeaceae
. J Ethnopharmac 1988;24:41-91.
Rajwar S, Khatri P, Patel R, Dwivedi S, Dwivedi A. An overview on potential herbal anticancer drugs. Intr J Res Pharm Chem 2011;1:202-10.
He W, Cik M, Van Puyvelde L, Van Dun J, Appendino G, Lesage A, et al.
Neurotrophic and antileukemic daphnane diterpenoids from Synaptolepis kirkii
. Bioorg Med Chem 2002;10:3245-55.
Ghosh S, Ahire M, Patil S, Jabgunde A, Bhat Dusane M, Joshi BN, et al.
Antidiabetic activity of Gnidia glauca
and Dioscorea bulbifera
: Potent amylase and glucosidase inhibitors. Evid Based Complement Alternat Med 2012;2012:929051.
Kraft C, Jenett-Siems K, Siems K, Jakupovic J, Mavi S, Bienzle U, et al. In vitro
antiplasmodial evaluation of medicinal plants from Zimbabwe. Phytother Res 2003;17:123-8.
Zhang SY, Zhang QH, Zhao W, Zhang X, Zhang Q, Bi YF, et al.
Isolation, characterization and cytotoxic activity of benzophenone glucopyranosides from mahkota dewa (Phaleria macrocarpa
(Scheff.) boerl). Bioorg Med Chem Lett 2012;22:6862-6.
Ibrahim N, Messay G, Berhanu T, Kissi M, Frehiwot T. Anti-Oxidant activity of 80% methanol extracts from Clerodendron myricoides, Satureja punctata, Urtica dioica, Ajuga remota
and Gnidia stenophylla
. Rev Cenic Cienc Biol 2010;41:1-7.
Mothana RA, Lindequist U. Antimicrobial activity of some medicinal plants of the Island Soqotra. J Ethnopharmacol 2005;96:177-81.
McGaw LJ, Van der Merwe D, Eloff JN.In vitro
anthelmintic, antibacterial and cytotoxic effects of extracts from plants used in South African Ethnoveterinary medicine. Vet J 2007;173:366-72.
Ndhlala AR, Finnie JF, Van Staden J. Plant composition, pharmacological properties and mutagenic evaluation of a commercial Zulu herbal mixture: Imbiza ephuzwato. J Ethnopharmacol 2011;133:663-74.
Clarkson C, Maharaj VJ, Crouch NR, Grace OM, Pillay P, Matsabisa MG, et al. In vitro
antiplasmodial activity of medicinal plants native to or naturalised in South Africa. J Ethnopharmacol 2004;92:177-91.
Bhandurge P, Rajarajeshwari N, Ganapaty S, Pattanshetti S. The Gnidia
genus: A review. Asian J Biomed Pharm Sci 2013;3:1-31.
Rushender R, Eerika M, Madhusudhanan N, Konda MV.In vitro
Antioxidant and free radical scavenging activity of Nympha eapubescens
. J Pharm Res 2012;5:3804-6.
Arnao M, Cano A, Acosta M. The hydrophilic and lipophilic contribution to total antioxidant activity. Food Chem 2001;73:239-44.
Patel DS, Shah PB, Managoli NB. Evaluation of in vitro
anti-oxidant and free radical scavenging activities of Withania somnifera
and Aloe vera
. Asian J Pharm Tech 2012;2:143-7.
Modi HK, Patel VM, Shrimanker MV, Patel KP.In vivo
antioxidant activity of extracts of Aegle marmelos
leaves. Am J Pharm Tech Res 2012;2:835-46.
Nethravathi HR, Kekuda TR, Vinayaka KS, Thippeswamy NB, Sudharshan SJ. Studies on antioxidant and anthelmintic activity of Gnidia glauca
(Fresen) Gilg. Asian J Biol Sci 2010;5:6-9.
Gupta A, Oza G, Durve A, Sharon M. Bactericidal effect of crude extracts of an endangered plant: Lasiosiphon eriocephalus
Decne. J Microbiol Biotech Res 2012;2:866-70.
Tiwary BK, Bihani S, Kumar A, Chakraborty R, Ghosh R. The in vitro
cytotoxic activity of ethno-pharmacological important plants of Darjeeling district of West Bengal against different human cancer cell lines. BMC Complement Altern Med 2015;15:22.
Tiwari PA. Review on traditional plants and herbs are used to evaluation for their anticancer potential. J Pharm Res 2017;11:547-53.
Kooti W, Servatyari K, Behzadifar M, Asadi-Samani M, Sadeghi F, Nouri B, et al.
Effective medicinal plant in cancer treatment, part 2: Review study. J Evid Based Complementary Altern Med 2017;22:982-95.
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