The fruits of
Methotrexate (MTX) or
Pharmaceuticals and nutritional sciences have recently witnessed the emergence of a new research field studying the impact of nutrition on human and animal health. At the present time, considerable attention is being paid to functional foods, which, in principle, apart from their classical notion of ‘adequate nutrition’, that is, a diet that provides basic nutritional functions, is being gradually replaced by ‘functional nutrition’; i.e., the food components have the potential to improve general physiological functions and reduce the risk of developing chronic diseases. There has been a virtual explosion of interest in identifying foods, food extracts and phytochemical formulations from plant sources that are able to mitigate oxidative and inflammatory stress (
The anti-inflammatory, antitumor and chemoprotective efficacy of the fruit extract of
MTX, silymarin (SLM), 2,2-diphenyl 1 picrylhydrazyl (DPPH), ascorbic acid, deoxy-ribose, ethylene-diamine, tetra-acetic acid (EDTA), sodium cyanide (NaCN), p-nitroblue tetrazolium chloride (NBT), riboflavin, eosin and α-tocopherol were purchased from Sigma-Aldrich; Merck KGaA. Riboflavin, sodium nitroprusside (SNP), sulphanilamide, trichloroacetic acid (TCA) and thiobarbituric acid (TBA) were purchased from HiMedia Laboratories Pvt. Ltd. Sodium dodecyl sulfate (SDS) and Harris hematoxylin were purchased from Spectrum Reagents and Chemicals Pvt. Ltd. Glutathione and 5-dithiobis-2-nitrobenzoic acid (DTNB; Ellmans reagent) were obtained from Molecular Probes Life Technologies (USA). Drabkin's reagent was purchased from Agappe Diagnostics Ltd. The creatinine, urea, serum glutamate-pyruvate transaminase (SGPT), alkaline phosphatase (ALP) and serum glutamic oxaloacetic transaminase (SGOT) analysis kits were purchased from Coral Clinical Systems. The highly specific sandwich ELISA kits for quantifying mouse IL-1β, TNF- α and IL-6 levels were purchased from PeproTech, Inc. All other chemicals used for experimental purposes were of analytical grade.
The fresh fruits of
The collected fresh fruits of
The FPD thus obtained was evaluated for determining the occurrence of various phytochemical constituents, such as flavonoids, terpenoids, glycosides, alkaloids, phenols, tannins, saponins, xanthoproteins and quinones using the following simple qualitative test (
HPTLC was performed on HPTLC plate's silica gel 60F 254, 7.0X10.0 cm plates (Merck KGaA), with toluene:ethyl acetate:methanol:formic acid (7:3:1:0.5) as a mobile phase. A CAMAG automatic TLC sampler 4 (ATS4) 170206 was equipped with a 25 µl syringe. A CAMAG TLC scanner 160623 was used for detection purposes. WIN CATS planar chromatography software was used to densitometrically quantify the bands. After HPTLC plate development, the TLC plates were dried and the chromatogram was visualized under 254 and 366 nm, and visible region using CAMAG TLC scanner 3.
A total of 28 polyphenols (catechin, quinine, catechol, epigallocatechin, tocopherol, naringenin, chlorogenic acid, gallic acid, syringic acid, epicatechin, caffeic acid, vanillic acid, ferulic acid, quercetin, myricetin, p-coumaric acid, apigenin, luteolin, kaempferol, rutin, shikimic acid, morin, diadzein, hesperetin, ellagic acid, cinnamic acid, genistein and chrysin) were quantitatively analyzed using the LC-MS/MS system (Nexera with LCMS-8045 (Shimadzu Corporation)-HPLC (Nexera LC-30 AD). It was coupled with an autosampler (SIL-30AC), prominence diode array detector (SPD-M20), temperature-controlled column oven (CTO-20AC) and a triple quadrupole mass spectrometer (Nexera with LCMS-8045 (Shimadzu Corporation). Working standards (0.01-1 µg/ml) were prepared by diluting the stock solution with water. The quantification of all polyphenols was performed on a Shimadzu Shim-pack GISS C18 column (150x2.1 mm), with solvent A [water/formic acid (100/0.1%)] and solvent B (100% methanol) as a mobile phase. A linear gradient elution system was used to elute the polyphenols. The detailed analytical procedure was as follows: 5% of solvent B (0.5 to 1.9 min), 98% of solvent B (2.0 to 10.0 min), 98% of solvent B (10.1 to 15 min) and 5% of solvent B (15.1 to 17 min). The flow rate was 0.3 ml/min with an injection volume of 10 µl, and an oven temperature of 40˚C. The detection of polyphenols was performed in multiple reaction monitoring (MRM) mode using LC-MS/MS with positive, and negative modes of ion switching electrospray ionization (ESI) method. LC-MS/MS data acquisition and processing were performed using Lab Solutions Software 5.6 (Shimadzu Corporation). Each calibration solution was performed in triplicate, and the results obtained are expressed as µg/g with SD (n=3).
The DPPH free radical scavenging activity of the FPD was estimated by the method described in the study by Magalhaes
The superoxide scavenging activity was determined by the method previously described by McCord and Fridovich, with minor modifications (
The iron (Fe3+)-ascorbate-EDTA-hydrogen peroxide (H2O2) system (Fenton reaction) was employed to assay the hydroxyl radical scavenging activity by the methods described in the studies by Kunchandy and Rao (
The nitric oxide (NO) radical scavenging activity of FPD was determined by the procedure described by Green
The capability of FPD to scavenge radical was calculated using the following formula: Percentage inhibition of radical=[A0-A1/A0] x100, where A0 was the OD of the control, and A1 was the OD of the sample/standard. IC50 values were calculated using software program Easy Plot (Epw.32).
BALB/c mice (6-8 weeks old, weighing 22-25 g) were purchased from the Sree Chitra Thirunal Institute for Medical Sciences and Technology (SCTIMST), Thiruvananthapuram, India. The animals were acclimatized for 1 month prior to the study. The experimental animals were housed under standard animal housing conditions (25±2˚C, 50% relative humidity and 12-h light/dark cycle) and were provided with rodent chow (VRK Nutritional Solutions, Maharashtra, India), and tap water
Following acclimatization under laboratory conditions, the BALB/c mice (n=24) were randomly divided into 4 groups (n=6). Group 1 was served as the sham-operated (sham) control. Group 2 served as the MTX control and was intraperitoneally administered with a single dose of MTX [20 mg/kg body weight (BW), intraperitoneally (i.p.)] on day 5 (
Changes in average BW of all the animals were measured at the beginning (day 0) prior to FPD administration, and then every 3 days thereafter, continuing throughout the entire experimental period (i.e., up to 10 days). Following euthanization, relative organ weight, and blood parameters were determined. The vital organs of experimental animals were excised immediately, washed thoroughly using PBS (pH 7.4). The data for organ weight are expressed as the relative organ weight (absolute organ weight/BW of the mice x100). Blood was collected via cardiac puncture immediately following euthanasia, and hematological parameters, such as Hb content, and total leukocyte count (WBC count) were also determined.
All animals were euthanized on the 11th day of the experiment by cervical dislocation. Separated serum was used for estimating hepatic and renal toxicity marker enzymes, such as SGOT, SGPT, bilirubin, ALP, urea and creatinine, measured using standard diagnostic kits from Coral Clinical Systems.
All animals were euthanized on the 11th day of the experiment by cervical dislocation. Liver, lung and kidney tissue samples were dissected out, washed with physiological saline, minced and homogenized in 25% (w/v) ice-cold 0.1 M Tris buffer (pH 7.4) using a homogenizer, and the resulting homogenate was then used for estimating the level of antioxidant enzymes and oxidative stress markers, such as catalase, GPx, SOD, GSH, MDA and NO (
A portion of the liver, kidney and lungs from each group was excised and fixed in 10% formalin. The preserved samples were then processed for routine paraffin wax block preparation. Following several steps of dehydration with alcohol i.e., 70% alcohol for 1 h at 37˚C, 80% alcohol for 1 h at 37˚C, 90% alcohol for 1 h at 37˚C and 100% alcohol for 2 h at 37˚C. The tissue specimens were then incubated with xylene for 2 h at 37˚C. The specimens were then embedded in paraffin blocks and 4-µm-thick sections of samples were prepared using a Weswox optic rotary microtome, MT-1090. A and then transferred the tissue section into a clean glass slide. The slide was then heat-fixed at 60˚C for 1 h and then incubated in xylene for 10 min at 37˚C. This was followed by rehydration using 100% alcohol for 1 h at 37˚C, 90% alcohol for 1 h at 37˚C, 80% alcohol for 1 h at 37˚C and 70% alcohol for 2 h at 37˚C. The tissue specimen was then rinsed with water. The tissue specimen was then stained with hematoxylin for 2 min at 37˚C, then rinsed with water for 1 min at 37˚C, then dipped the slide in acid alcohol solution for removing excess stain, and rinsed again with water for 1 min at 37˚C. This was followed by immersion of the slide in 0.3% ammonium hydroxide solution for 1 min, and rinsing again with water for 1 min at 37˚C, and further immersion in eosin for 45 sec, washing with water for 1 min, and dehydration using 70% alcohol for 1 h at 37˚C, 80% alcohol for 1 h at 37˚C, 90% alcohol for 1 h at 37˚C and 100% alcohol for 2 h at 37˚C. The mounted specimens were analyzed and examined under Olympus CKX53 microscope (Olympus Corporation) by a certified pathologist. A total of three sections/mouse was analyzed, and random sections on each slide were used for analysis.
On day 11, blood was collected by cardiac puncture, and serum was separated by centrifugation (2,000 x g) for 10 min at 4˚C. The serum was then used for quantifying the level of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) using highly specific sandwich ELISA kits as per the manufacturer's instructions (PeproTech, Inc.).
Values were expressed as the mean ± standard deviation (SD). The significant result difference between the groups was determined using one-way ANOVA followed by Dunnett's test using Instat version 3.0 software (GraphPad Software Inc.). A P-value <0.05 was considered to indicate a statistically significant difference.
The phytochemical screening of FPD revealed the presence of bioactive components, such as flavonoids, terpenoids, glycosides, alkaloids, phenols, tannins, xanthoprotiens and saponins. Quinones were found to be absent in FPD. The results are presented in
HPTLC analysis of FPD revealed several peaks of phytocompounds. The obtained retardation factor (Rf) values of the different peaks are summarized in
LC-MS/MS chromatograms of the standards and extract, and polyphenols identified in FPD are depicted in
The results of the DPPH scavenging activity of FPD are shown in
The hydroxyl radical scavenging activity of various concentrations of FPD was compared with the same concentrations of the standard (α-tocopherol) ranging from 20 to 400 µg/ml, and is depicted in
The results of the superoxide scavenging activity of FPD are depicted in
The results of the NO scavenging activity of FPD are shown in
The anti-lipid peroxidation efficacy of different concentrations of FPD was compared with the same concentrations of standard (ascorbic acid) ranging from 20 to 400 µg/ml and is shown in
The effects of different treatment regimens on the total WBC count are depicted in
The effects of the different treatment regimens on the Hb content are presented in
A schematic of the effects of the different treatment regimens on BW is shown in
Changes in the relative organ weights of the spleen, thymus, liver, kidney and lungs are depicted in
The effects of FPD on serum hepatic (SGPT, SGOT, bilirubin and ALP) and renal (urea and creatinine) toxicity marker levels are presented in
The effects of treatment regimens on liver, kidney, and lungs oxidative parameters are illustrated in
The effects of FPD on MTX-induced histopathological changes in the liver, kidney and lung tissue samples are illustrated in
The administration of a high dose of MTX markedly elevated the serum cytokine levels in the experimental mice. However, treatment with FPD along with MTX reduced the levels of serum pro-inflammatory cytokines (
Preliminary qualitative phytochemical analysis of FPD revealed the presence of diverse phytochemical components, which includes flavonoids, terpenoids, glycosides, alkaloids, phenols, tannins, saponins and xanthoproteins, and has been reported to possess anti-inflammatory, antioxidant, antiparasitic, antimicrobial and anticancer activity. Over the past few years, flavonoids have elicited increasing attention due to their distinct potential health benefits (
In the present study, LC-MS/MS analysis of FPD revealed the presence of several flavonoids and phenolic compounds, including naringenin, quercetin, catechin, tocopherol, quinine, gallic acid, epicatechin, caffeic acid, epigallocatechin, myricetin, p-coumaric acid, luteolin, apigenin, kaempferol, rutin, cinnamic acid, hesperetin, shikimic acid and genistein, which have been reported to possess potent antioxidant properties (
The free radical scavenging activity of antioxidants on DPPH is considered to be due to their hydrogen-donating property (
Superoxide anion radicals are highly unstable free radicals mainly produced by activated phagocytes, such as eosinophils, neutrophils, monocytes, and macrophages. Cells have developed an elaborate complex system of enzymatic and non-enzymatic antioxidant defense mechanisms, which can counteract these indigenously developed ROS. SOD is one such key powerful detoxification enzyme found in almost all living organisms and is responsible for protecting cells from oxidative-stress induced cell death. The excessive generation of free radicals disrupts homeostasis between oxidants and antioxidants, invariably accompanies cellular damage (
Among ROS, hydroxyl radicals are the most potent, extremely reactive, highly lethal and damaging oxidants that can interact with chromatin; exposure to these radicals causes extensive damage, which include a wide range of sugar- and base-derived products, protein fragmentation, DNA-protein cross-links and DNA strand breaks (
It has been long been recognized that generations of free radicals can inflict the oxidative deterioration of polyunsaturated fatty acids (PUFAs) (
Reactive nitrogen species (RNS) and NO are highly unstable freely diffusible free radicals that readily attack chromatin leading to deamination of cytosine, adenine, and guanine. The toxicity induced by NO markedly increases by reacting with superoxide anion radical, forming an extremely reactive peroxynitrite radical (ONOO-). NO is a potent pleiotropic intracellular and intercellular signaling molecule having an impressive spectrum of diverse physiological and pathophysiological functions. NO can react rapidly with molecular oxygen to form nitrogen oxides (NO2, N3O4, and N2O4). The higher nitrogen oxides either interact with certain biomolecules such as amines, and thiols or otherwise hydrolyze to produce nitrite (NO2-), and nitrate (NO3-). Furthermore, NO can also interact rapidly with superoxide radical forming ONOO-. The degree of tissue damage is mainly persuaded by microenvironmental conditions under which NO is released. All these intermediators and progenitor products induce deamination in cytosine, adenine and guanine. Chronic exposure to NO radicals is associated with various types of disorders, including AIDS, juvenile diabetes, cancer, multiple sclerosis, arthritis, Alzheimer's and ulcerative colitis (
Furthermore, the
Hepatotoxicity is another most common complication associated with MTX treatment. An increment in the levels of serum SGOT, SGPT and ALP may be attributed to the damaged structural integrity of the hepatic membrane (possibly via lipid peroxidation and oxidative stress) (
The administration of MTX leads to the upregulation of nephrotoxicity marker enzymes (urea and creatinine), which ultimately culminate the disruption of glomerular apparatus (
It has been demonstrated that MTX administration significantly increases hepatocellular ROS and NO levels (
In conclusion, the present study demonstrates that FPD contains higher concentrations of tannins, flavonoids, phenolics, saponins and several other bioactive components. Furthermore, the results revealed that supplementation with FPD significantly prevented oxidative stress-associated tissue damage induced by MTX; FPD may have great potential for application in future disease therapeutic strategies. However, further studies are required to better ascertain and disseminate its pharmacological properties, which may provide aid in the discovery of novel drugs and the developmental arena.
The authors are thankful to Dr Rekha A. Nair, Director, Regional Cancer Centre (RCC) and Dr S. Kannan, Head, Division of Cancer Research, RCC for providing valuable support required for the study. The authors also acknowledge Dr Nisha Prakasan of CSIR-NIIST for assisting with the LC-MS/MS analysis.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
SSD was involved in the study methodology and investigation, as well as in the writing of the original draft, visualization, and data collection and interpretation. SD was involved in formal analysis and investigation. RPM was involved in data analysis and interpretation. CG was involved in the study conceptualization, in the writing, reviewing and editing of the manuscript, in study supervision, project administration, funding acquisition, and data analysis and interpretation. CG and SSD confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
The present study was approved by the Institutional Animal Ethics Committee (IAEC), Regional Cancer Centre (RCC), Thiruvananthapuram. (IAEC/RCC NO. 6/18).
Not applicable.
The authors declare that they have no competing interests.
HPTLC chromatogram of FPD. (A-a) HPTLC chromatogram of FPD at 254 nm. (A-b) Peak analysis of HPTLC chromatogram at 254 nm. (A-c) Peak area with Rf values of obtained chromatogram at 254 nm. (A-d) Image documentation of developed HPTLC plate at 254 nm. (B-a) HPTLC chromatogram of FPD at 366 nm. (B-b) Peak analysis of HPTLC chromatogram at 366 nm (B-c) Peak area with Rf values of obtained chromatogram at 366 nm. (B-d) Image documentation of developed HPTLC plate at 366 nm. (C-a) HPTLC chromatogram of FPD at visible range. (C-b) Peak analysis of HPTLC chromatogram at visible range. (C-c) Peak area with Rf values of obtained chromatogram at visible range. (C-d) Image documentation of developed HPTLC plate at visible range. HPTLC, High performance thin layer chromatography; FPD, fruit extract of
Polyphenolic profiling of FPD and its quantification using LC-MS/MS: Chromatogram of FPD. (A) LC-MS/MS chromatogram showing standard polyphenols. (B) LC-MS/MS chromatogram showing polyphenol concentration in FPD. (C) LC-MS/MS analysis of polyphenols in FPD. FPD, fruit extract of
Evaluation of
(A) Effect of FPD on the total leukocyte count (WBCs). (B) Effect of FPD on the Hb level. (C) Assessment of body weight of animals following the respective treatments. The body weight of each animal was monitored every 3rd day (starting from day 0, prior to the first treatment) and statistical analysis was performed. All values shown are the mean ± SD. *P<0.05 and **P<0.01, MTX alone vs. MTX + FPD or MTX + SLM. Hb, hemoglobin; MTX, methotrexate; SLM, silymarin; FPD, fruit extract of
Effects of FPD on liver, kidney and lung markers (A) NO; (B) MDA; (C) GSH; (D) SOD; (E) GPx and (F) catalase levels following the respective treatments. All values shown are the mean ± SD. *P<0.05 and **P<0.01, MTX alone vs. MTX + FPD or MTX + SLM. MTX, methotrexate; SLM, silymarin; FPD, fruit extract of
Protective effect of FPD on liver, kidney and lungs histology during MTX-induced toxicity. (A) Protective effect of FPD on liver histology during MTX-induced toxicity. (a) Section from normal mouse. (b) MTX alone group (arrows represent altered lobular architecture with inflammatory cell infiltration). (c) MTX + SLM group. (d) MTX + FPD group. (B) Photomicrographs of mouse kidney following the respective treatments. (a) Section from normal mouse. (b) MTX alone group (arrows represent focal epithelial loss and inflammatory cell infiltration). (c) MTX + SLM group. (dd) MTX + FPD group (C) Protective effect of FPD on lung histology during MTX-induced toxicity. (a) Section from normal mouse. (b) MTX alone group (arrows represent periportal inflammation, cytoplasmic vacuolation and scattered infiltration of lymphocytes) (c) MTX + SLM group. (d) MTX + FPD group. MTX, methotrexate; SLM, silymarin; FPD, fruit extract of
Pro-inflammatory cytokine (TNF-α, IL-1β and IL-6 and) profiling. Statistical analysis was performed using one-way ANOVA. All values were expressed as the mean ± SD. **P<0.01 compared with MTX alone. MTX, methotrexate; FPD, fruit extract of
Conceptual diagram of the antioxidant mechanisms of FPD. FPD, fruit extract of
Phytochemical components in FPD.
Sl. no. | Component | In FPD |
---|---|---|
1 | Flavonoids | + |
2 | Terpenoids | + |
3 | Glycosides | + |
4 | Alkaloids | + |
5 | Phenols | + |
6 | Tannins | + |
7 | Saponins | + |
8 | Xanthoproteins | + |
9 | Quinones | - |
FPD, fruit extract of
Effect of treatments on relative organ weights.
Relative organ weight (g/100 g body weight) | |||||
---|---|---|---|---|---|
Group | Spleen | Thymus | Liver | Kidney | Lungs |
Sham | 0.46±0.04 | 0.26±0.02 | 5.58±0.52 | 1.36±0.05 | 1.15±0.20 |
MTX | 0.30±0.02 | 0.10±0.02 | 4.40±0.34 | 1.29±0.10 | 0.80±0.05 |
MTX + SLM | 0.47±0.03 |
0.18±0.07 |
5.70±0.16 |
1.38±0.04 | 0.98±0.13 |
MTX + FPD | 0.41±0.05 |
0.21±0.02 |
5.31±0.26 |
1.33±0.07 |
1.08±0.10 |
All values are expressed as the mean ± SD.
aP<0.01 and
bP<0.05, compared with MTX alone. MTX, methotrexate; SLM, silymarin; FPD, fruit extract of
Effect of FPD on serum SGOT, SGPT, bilirubin and ALP levels in experimental animals treated with MTX.
Groups | SGOT (U/l) | SGPT (U/l) | Bilirubin (g/l) | ALP (U/l) |
---|---|---|---|---|
Sham | 11.99±3.80 | 39.88±6.23 | 1.06±0.26 | 3.88±0.54 |
MTX alone | 23.84±8.09 | 82.04±9.30 | 2.02±0.28 | 7.21±0.58 |
MTX + SLM | 14.19±3.24 |
41.33±5.13 |
1.19±0.24 |
4.05±0.65 |
MTX + FPD | 12.64±2.80 |
45.33±6.43 |
1.16±0.16 |
3.924±0.22 |
All values are expressed as the mean ± SD.
aP<0.01, compared with MTX alone. MTX, methotrexate; SLM, silymarin; FPD, fruit extract of
Effect of FPD on serum urea and creatinine levels in experimental animals treated with MTX.
Urea (g/l) | Creatinine (g/l) | |
---|---|---|
Sham | 1.39±0.09 | 2.91±0.28 |
MTX alone | 2.17±0.27 | 3.74±0.50 |
MTX + SLM | 1.55±0.27 |
3.03±0.09 |
MTX + FPD | 1.17±0.06 |
3.1±0.14 |
All values are expressed as the mean ± SD.
aP<0.01, compared with MTX alone. MTX, methotrexate; SLM, silymarin; FPD, fruit extract of