Cornsilk B.J.

It has been reported that consumption of corn silk has no adverse effects and that it is safe for human use. Previous studies have confirmed the presence of multiple bioactive compounds in corn silk, including proteins, polysaccharides 5 , flavonoids 6 , vitamins, tannins, alkaloids, mineral salts 7 and steroids. There have been numerous studies on the bioactivity of corn silk constituents, including its antitumor capacities 8 , anti-diabetic activity in hyperglycemia rats 5 and anti-fatigue activity 9. In our preliminary experiments, corn silk extract was noted to inhibit cell growth and trigger apoptosis 8.

However, the specific active ingredients in corn silk extract that induce cancer cell apoptosis remain unknown. In the present study we investigated whether corn silk extract could induce apoptosis through the mitochondrial pathway in LoVo human colon cancer cells, and whether it could affect cell proliferation, cell cycle progression and protein expression. Collected corn silk was extracted in hot water three times and filtered; the brown filtrate with most of the small soluble compounds was removed.

The rest of the corn silk was air-dried and then pulverized into a homogeneous size by a disintegrator. Then, the extract was obtained by centrifugation, filtering through a filter paper and cellulose ester membrane with 0. Corn silk extract, a light brown powder, was dissolved in Dulbecco's modified Eagle's medium DMEM , filtered through a 0.

The extraction of proteins was determined according to the Kjeldahl method 10 ; total sugar was determined by the phenol-sulfuric acid assay 11 and reducing sugar by the 3,5-dinitrosalicylic acid DNS assay The effects of corn silk extract on cell proliferation were evaluated by MTT assay. Following overnight growth, the culture medium was replaced with various concentrations 1. Thereafter, the absorbance was measured at nm using an enzyme-linked immunosorbent assay plate reader.

Cell survival percent was calculated using the following formula: The cells were incubated with RNase A 0. The cell cycle distribution and the percentage of apoptotic cells sub-G1 fraction were determined by flow cytometry Following ethidium bromide staining, images of the DNA ladders were captured under UV as previously described Following treatment with various concentrations 5. Following incubation, the cells were washed twice with PBS and analyzed by flow cytometry as previously described 16 , The cells were harvested and extracted using a nuclear and cytoplasmic extraction kit.

Then, the proteins were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The separated proteins were transferred to polyvinylidene difluoride membranes and blocked with bovine serum albumin blocking buffer for 2 h at room temperature. On the second day, the membranes were incubated with the relevant secondary antibodies 1: Finally, the transferred proteins were visualized using diaminobenzidine as previously described 18 , Statistical differences between corn silk extract-treated and control groups were evaluated using Student's t-test with SPSS As shown in Table I , corn silk extract contained In a previous study, Ooi and Liu observed that the anticancer activity of polysaccharides may be a consequence of the stimulation of cell-mediated immune response In another study, Yang et al demonstrated that corn silk polysaccharides not only inhibited the tumor growth, but also extended the survival time of H22 tumor-bearing mice Food proteins may be considered as a source of nutraceutical peptides and amino acids that exert biological functions to promote health and prevent disease, including cancer As shown in Fig.

Compared with the MGC cells and HT cells, LoVo cells were more sensitive to corn silk extract and thus were used to explore the related mechanism of corn silk extract-induced apoptosis in LoVo cells. The cells were exposed to various concentrations of corn silk extract for 24, 48 and 72 h. PI staining was used to detect the effects of corn silk extract at various concentrations on colon cancer cells.

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Forty-eight hours after treatment, a sub-diploid peak was present in the left part of the graph. With the increase in corn silk extract concentration, the sub-diploid peak became increasingly evident, representing a gradient of mortality. These results reveal that corn silk extract has a significant inhibitory effect on cancer cell growth. Moreover, the sub-G1 fraction was increased in a drug concentration-dependent manner. The number of S-phase cells was The data revealed that the relative proportion of cells in the S phase increased when the concentration of corn silk extract was increased.

Correspondingly, cells in the sub-G1 phase were also increased in a drug concentration-dependent manner, which was consistent with the flow cytometry data. Therefore, the cell cycle inhibitory effect of the corn silk extract mainly involved S-phase arrest. Effects of corn silk extract on cell cycle distribution.

The histograms reveal the percentage of cells in the indicated phases of the cycle. E The graph reveals the distribution of cells in different phases of the cell cycle by flow cytometry in LoVo cells, and demonstrates that corn silk extract arrests cells in the S phase. We observed that, in contrast to the control group, treatment of LoVo cells with corn silk extract for 48 h resulted in internucleosomal DNA fragmentation consisting of — bp, a characteristic of apoptosis, exhibiting a typical ladder-like pattern of degraded DNA products on agarose gel electrophoresis.

A previous study reported that in the event of apoptosis, DNA gel electrophoresis exhibited a clear ladder. DNA gel electrophoresis is considered one of the gold standards in the determination of apoptosis As illustrated in Fig. In the mitochondrial apoptosis pathway, the decrease in mitochondrial membrane potential is an early event in apoptosis.

It was reported that the proteins extracted from corn silk could decrease mitochondrial membrane potential 22 , which is in accordance with our results. However, in the low-dose group, the mitochondrial membrane potential was increased compared with other corn silk extract-treated groups, which may be related to the selection of cells.

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The elevation of calcium ion levels is associated with signal transduction in the early stages of apoptosis. Mitochondria represent one of the intracellular calcium pools. This process was synergized by other pro-apoptotic factors leading to apoptosis of a large number of cells.

Using western blot assay, we have shown above that apoptotic pathway induction by corn silk extract treatment was mediated by affecting the Bax and Bcl-2 levels, causing dysfunction of mitochondria, promoting the release of cytochrome c and activating caspase-9 and caspase-3 15 , Effects of corn silk extract on apoptosis-associated proteins in LoVo cells.

Cells were treated with various concentrations of corn silk extract for 48 h, then apoptosis-associated proteins were examined by western blot analysis as described in Materials and methods. Bcl-2, B-cell lymphoma 2. Western blot analysis data are presented as the relative expression of selected proteins compared with GAPDH. Caspase is a family of proteases that plays a significant role in the process of apoptosis.

Caspase-9 is involved in the upstream stages of the signal transduction cascade of apoptosis. Following the release of cytochrome c from mitochondria, caspase-9 is activated to form a complex with cytochrome c. The activation of caspase-9 then activates caspase-3, thereby facilitating subsequent apoptotic signaling. Caspase-3 is a key enzyme in the apoptotic cascade, playing an essential role in chromatin condensation, DNA fragmentation and other nuclear apoptotic processes This further supports the hypothesis that corn silk extract significantly induced apoptosis in LoVo cells.

Bcl-2 family proteins also play crucial roles in apoptosis.

This family consists of two types of proteins: Determination of SOD activity from kidney supernatant: Determination of kidney index: The kidneys of each animal were fixed in buffered formalin. Kidneys were processed and embedded in paraffin wax. Three micro mitter thick paraffin sections were stained with Haematoxylin and Eosin for light microscope examination. Effect of the extract on renal function improvement in gentamicin-piroxicam induced renal failure: Effect on creatinine serum levels and histopathological cross section after 7 consecutive day administration of gentamicin-piroxicam: The administration of gentamicin and piroxicam for 7 consecutive days to each of tested group could increase creatinine concentration fold significantly at the first week week 1 when compared to the negative control group Fig.

There were no significant differences among the induced groups. The findings on the biochemical parameters were also supported by the histopathological results which were showing changes in kidney structure profile both in the cortex and medulla Fig. It was clearly found any tubular degeneration, glomerular atrophy, vacuole formation and hematuria in several places in the cross section of groups induced. Enhancement of creatinine concentration followed by more severe damage that can be observed on microscopic section.

Effect of extract on serum creatinine levels: At week 2, as general, each of induced group include positive control group experienced reduction of creatinine concentration it is predicted because of the body homeostasis. However, creatinine concentration of the positive control group was higher and significantly different compared to the test group and the negative control group. Each of the test group, either single or in combination, experienced reduction creatinine concentration which was significantly different compared to the positive control group and the baseline value of each group.

There were no significant differences among the test groups. From these data, it could be seen that the extract in half dose combination could produce effects more or less comparable to the single form of extract. Administration in one dose combination did not provide any significant better activity than single extract and half dose combination.

This could be seen clearly in Fig. Effect of extract on serum urea levels: Similar profiles were also found on the second parameter, serum urea level Fig. Serum urea levels of positive control group were decreased in the following weeks, but tend to increase at week 5. The tendency was not found in the test groups which were given extract. Effect of leaves extract on renal histopathological: Although improvement was also observed in the positive control group, each of microscopic cross sectional of the test group exhibited better performance and started to approach negative control group, especially in the medulla part Fig.

In this study, there was a finding about an increase in the amount of TBARS in group induced by gentamicin and piroxicam. Each test group which was given extract had less amount of TBARS than and significantly different from the positive control group Fig. Effect of extract on catalase activity: Positive control group recorded a significantly lower level of catalase compared to the negative control group.

The group which was treated with extract recorded significantly elevated levels of catalase indicating restoration of levels of catalase closer to normal, untreated animals Fig. Effect of extract on SOD activity: Group induced kidney failure was found to have decrease SOD activity.

Treatment with extract produced significant increase in these enzyme levels Fig. Effect of extract on organ index: The positive control group had the highest renal index among the other groups and was significantly different compared to the negative control group. The extract group had lower renal index compared to positive control group and there were no differences among the test groups Fig.

The results which were shown by enhancement of creatinine and urea concentration in the first week of treatment, supported previous research that suggested that rat models of kidney failure could be formed rapidly by giving a combination of gentamicin and piroxicam Sukandar et al. The doses were given for 7 days for the duration had been reported to induce kidney damage. Enhancement of creatinine levels indicated that the inductor, especially gentamicin, could cause a decrease in glomerular filtration function, whereas, the histological results showed that gentamicin give a major influence on tubule, especially the proximal tubule Rybak et al.

Tubular damage was probably derived as a result of the apoptosis which is essentially one of the important processes to maintain homeostasis. In that process, both lysosomes and mitochondria sends signals that cause the rupture of membranes and release of lysosomal acid hydrolases De Souza et al. Therefore, improvement of kidney function could be achieved by prolong the application period rather than increasing the dose. The increasing level of urea was clearly seen in the fifth week of therapy which was confirmed the severe level of kidney damage Parlakpinar et al.

The increasing indicated that the damage was likely to remain in the positive control group. The elevation tendency was not found in the test groups which were given extracts. This result confirmed the kidney protective activity provided by the extract.

Pro-apoptotic and anti-proliferative effects of corn silk extract on human colon cancer cell lines

Kidney damage was also supported by the values of organ index which were higher compared to the negative control group. The high values index of the positive control group and the test group might be due to the occurrence of edema and fluid accumulation in the pathological extravasal space, especially within the interstitium, due to tubular necrosis Meliani, ; Zulkarnain, and might also be caused by the proliferation and apoptosis of mesangial cells simultaneously.

These mesangial cells are special cells which are located in around blood vessels in the kidneys. In one study, either in vivo or in vitro way, it was revealed that gentamicin stimulated mesangial cell contraction and proliferation. Gentamicin was also able to increase the expression or activation of the proapoptosis protein so that, it could cause rupture of the lysosomal membrane and release of acid hydrolases that contributed to apoptosis and necrosis of proximal tubular cells De Souza et al.

However, the extract was able to show improvement in biochemical and histological parameters so, it was possible that the extract could maximize the performance of mesangial cells which were still functioning. Other parameters which are analyzed in this study are related to oxidative stress. Oxidative stress is an abnormal condition in which increased oxidant product could not be opposed by increased production of antioxidants. The absence makes the body's equilibrium are not in a safe zone Rico et al.

Various studies have shown the involvement of oxidative stress in many of degenerative diseases, one of which is kidney failure Polat et al. Uremia itself causes increased production of free radical s and even hemodialysis procedure could sometimes increase the amount of radicals Galle, These things cause higher risk of morbidity and probability of suffering other diseases of the kidney failure patient compared to common patients without kidney failure Luciak, There are several parameters that can be used to predict the level of oxidative stress in patients with kidney failure, such as conjugated diene, antioxidant enzymes, peroxidation products of metilguanidin as creatinine, serum antioxidant activity and lipid peroxidation Gotoh et al.

It was found that oxygen radical species, such as superoxide anion, hydrogen peroxide and hydroxyl radicals, took a role in the pathophysiology of gentamicin. Oxygen radical species causes rapid changes in the composition of the membrane lipids or better known as lipid peroxidation. Furthermore, the cells quickly lose their osmotic balance resulting in increased intracellular calcium levels.

On the other hand, radical species can consume most of the performance of the enzyme, such as Superoxide Dismutase SOD and catalase, thereby reducing the activity of these enzymes Abdel-Raheem et al. Lipid peroxidation could be defined as oxidative damage to lipid structures that contain double bonds between carbons.

Another definition reveals that lipid peroxidation is a process associated with free radical s, a process that is not controlled and can run continuously causing disruption on the membrane, lipids and other cell components. Lipid peroxidation that occurs continuously can be a major factor in the pathogenesis of complications of kidney failure Shanmugam et al. As stated in the previous paragraph, in the human body, the structure of the lipids are commonly found in cell membranes. That makes it more complex is that the membrane becomes first defense for each of the cell, such as mitochondria, plasma, endoplasmic reticulum, lysosomes, peroxisomes and others.

Additionally metabolites formed between the oxidation processes can give adverse effects on other places than the place of origin oxidation Devasagayam et al. One result of lipid peroxidation is aldehydes compounds that can react with the acid to form a pink thiobarbiturat which are easily detected using a spectrophotometer.

Such compounds are often referred to as acid reactive compounds tiobarbiturat or TBARS thiobarbit uric acid reactive substances. TBARS as one of the products of lipid peroxidation can be used as a reference for predicting how many are the radicals production.

It appears that kidney failure tends to weaken the defense system against free radicals. This makes the high amount of free radical s that have an impact on the high levels of tissue damage. These facts confirm the microscopic cross section results which are showing higher levels of tubular necrosis in the positive control group than the test group or the negative control group. Much evidence suggested that the excessive amount of free radical s due to kidney failure may worsen the disease and increase the risk of complications.

From these results, it appears likely that the extract has antioxidant activity because it can decrease the level of lipid peroxidation in the test group. The decline may be caused by the activity of membrane lipid protection, repair tissue that is provided by the extracts, or free radical damping which reduce directly amount of free radicals found so that there are not radical that can attack the lipid membranes. Furthermore, these results corroborate previous data showing that administration of combination do not give superior activity rather than single extract use Prasanna and Purnima, It shows that the extract can improve the resistance of the cell membrane against oxidant so that tissue damage can be prevented or corrected.

Higher level of resistance cause lower serum creatinine concentration of the test group compared to its initial value and can mimic serum creatinine of negative control group. Protection system against oxygen radical species or oxidation products of lipids, proteins and DNA is provided by antioxidant enzymes such as SOD and catalase. In other words, SOD and catalase are the two main examples of radical fighting enzymes in vivo. It was found that there was decreased activity of both of enzymes, SOD and catalase, in kidney failure condition.

Pro-apoptotic and anti-proliferative effects of corn silk extract on human colon cancer cell lines

As the result, there was increased number of superoxide anion and hydrogen peroxide to produce hydroxyl radicals. In the end, the hydroxyl radical can initiate lipid peroxidation.

This process can increase the level of damage to the kidneys Palani et al. SOD can catalyze dismutase of superoxide anions into hydrogen peroxide which are then deactivated by catalase into water Kim et al. This indicates that in a state of oxidative stress , there was decreased activity or inactivation of the antioxidant defense system Olagunju et al. Decreased activity of the enzyme sometimes is a compensation for the increased number of radicals. In other words, an increase in the number of radical forces the immune system to issue more work to eliminate the oxidant.

However, it can trigger a series of other, more harmful effects Geo Vigila and Baskaran, The extract, either alone or in combination, can increase the activity of SOD and catalase enzyme compared to the positive control group Fig. The results of this study confirmed that administration of inductors, gentamicin and piroxicam, could increase lipid peroxidation in the kidney that was characterized by increased TBARS and decreased antioxidant enzyme activity although other damage mechanisms may occur as well. There was a tendency that either corn silk or binahong extracts have antioxidant activity that could protect kidney from nephrotoxicity.

Antioxidant agents have been shown to have the ability to prevent or repair damage to the kidneys. The above data indicated that the extract can help improve antioxidant status in rat model of kidney failure. These data confirm the results of previous studies related to corn silk. Various studies have revealed the antioxidant activity of the corn silk which may be caused by the high content of flavonoid or phenolic compounds Ebrahimzadeh et al.

In connection with its antioxidant extract, corn silk extract are also potentially be used to treat other diseases associated with oxidative stress. While binahong leaves, not many research publications that reveal about the activities associated with antioxidants. Some studies reveal binahong activity as antibacterial and wound healing Astuti et al.

However, it is possible that binahong also have antioxidant activity given the flavonoid contained in the extract. Flavonoids are phenolic compounds found in many plants. Plants used to use flavonoids to protect themselves against oxidative damage by inhibiting or reduce free radical s and reactive oxygen species that arise due to sun exposure.

It is caused by the presence of conjugated ring structures and hydroxyl groups. Therefore, it is suggested that high flavonoids have a role in preventing oxygen radical species to cause cytotoxicity and tissue damage in humans Anila and Vijayalakshmi, Suppression activity against oxidative stress may also be generated by the interaction among components contained in the extract. In this case, CCl4 produce damage through free radical production so, there are similarities that can be rationalized on the conditions of this study Maiza-Benabdesselam et al.

It is also predicted that the presence of tannin substances in corn silk may provide protection against damage. Tannin substances have astringent like activities that can cause precipitation of proteins in the cell membrane which form the barrier that prevents the attack by free radical s. Both silk of corn Zea mays L. Steenis leaves extracts could improve kidney function in rat model of kidney failure.

Combination of a half dose of each extract showed effects comparable or slightly better than an individual extract showing to have at least additive effect. Reduction of oxidative stress provided by each extracts and their combinations might be correlated with mechanism of repairing kidney failure. Green tea ameliorates renal oxidative damage induced by gentamicin in rats.

Evaluation of antioxidant and antibacterial activities of egyptian Maydis stigma Zea mays hairs rich in some bioactive constituents. Antioxidant action of flavonoids from Mangiferra indica and Emblica officinalis in hypercholesterlomic rats.

Determination of saponin compound from Anredera cordifolia Ten. Phytochemical constituents and antioxidant activity of various extracts of corn silk Zea mays L.