ACUTE ORAL TOXICITY
The present study evaluates the acute toxicity study of methanolic extracts of green grapes and black grapes. Although humans have ingested both green and black grapes for centuries and are continuing to do so without reported adverse effects, the current toxicology study presents somewhat little formal evidence regarding their safety.
In present study mice were divided into three groups with three mice per group. First group was kept as control i.e. mice of first group were given only distilled water orally while second and third group’s mice were administered orally with methanolic extracts of black grapes and green grapes respectively. As the accessible literature on Vitis vinifera L. reveals that it is nontoxic and mortality is unlikely at highest starting dose level (2000mg/kg body weight), so according to OECD 423 guidelines limit test was conducted deciding the dose based on 2000mg/kg body weight.
According to the results methanolic extracts of both green and black grapes were well tolerated by mice and they did not produce any kind of adverse effects and no mortalitily was recorded at a dose level as high as 2000mg/kg. No other changes in morphological appearance, body weight or food consumption was found. As far as control group is concerned, no changes were seen in mice belonging to that group too.
Present study can be correlated with the study of Geetha S. and Devaraj A. (2014) on acute toxicity of organic grapes (Vitis vinifera L.). In their study they proved methanolic extract of Vitis vinifera L. fruits as safe and non toxic to be administered orally.
Zeghad N. et al. (2016) also assessed acute oral toxicity of Vitis vinifera L. Fruit’s exract and acknowledged it toxicologically safe without any adverse effects at a high dose level up to 1000gm/kg. Likewise, in a study conducted by Bhaumik A. et al. (2015) ethanolic extracts of black grapes showed no adverse effects on WISTAR rats when administered orally at a dose level of 2000mg/kg.
Similarly, a dietary concentration of 2.0-2.5% Grape Seed Extract and Grape Skin Extract has been proved to be toxicologically safe in the studies conducted by Yamakoshi J. et al. (2002), Wren Allison F. et al. (2002), Bentivegna S.S. and Whitney K.M. (2002). Similar results were documented in several parts of the plant of Vitis vinifera L. Sharma Surendra K.R. et al. (2012) and Suralkar A.A. et al. (2015) reported non toxic properties of ethanolic extracts of Vitis vinifera L. roots and its leaves in their studies respectively.
An assessment report on Vitis vinifera L., folium by European Medicines Agency (2010) states that vine leaf aqueous extract had no toxicological adverse effect when tested upon WISTAR rats as well as on CFI mice. Draksh (Fruits of Vitis vinifera L.) failed to show any signs of adversity or toxicity up to 2000mg/kg body weight in the studies of Hadaginhal R.V. et al. (2011) and Kumar D. et al. (2009).
Kanagarla N.S.S.A.V. et al. (2013) studied that Vitis vinifera L. and its bioactive components have many pharmacological properties and their use does not cause any kind of harmful effect. Moreover, consumption of grape seed extract at the levels of up to 2.0% doesn’t induce any significant toxicological effects. Similar results were obtained in the study of Singh J. et al. (2009) where methanolic extracts of Vitis vinifera L. leaves didn’t show any adverse effect up to maximum dose level of 2000gm/kg of body weight.
In present study acute toxicity was evaluated by ‘Acute toxic class methods (OECD guidelines-423)’. During the surveillance period of 14 days, no significant toxicity occurred. No mortality with minute non-considerable behavioural changes was observed. So, the No-Observed-Adverse-Effect-Level (NOAEL) of methanolic extracts of both green and black grapes was recorded 2000mg/kg. Even a dose higher than 2000mg/kg is expected to be non toxic and safe. This indicates a lack of toxicity and supports the use of both extracts for further pharmacological and biological investigations.
In present study antitumor promoting activities of Methanolic green grape extract, Methanolic black grape extract and Methanolic green + black grape extract was examined in Swiss albino mouse skin epidermis. In particular, the ability of these extracts to inhibit DMBA and Croton oil induced skin carcinogenesis was evaluated. Three week post topical DMBA application, daily oral treatment of mice with above mentioned extracts was carried out. After 16 weeks, mice from all groups including control group were sacrificed for the analysis of tumor yield, tumor burden, tumor incidence and tumor diameter. During the entire procedure body weight of all mice was monitored weekly.
Significant reduction in tumor incidences was observed in the Methanolic green grape, Methanolic black grape and Methanolic green + black grape extract treated experimental groups (83.3, 50 and 50 % in groups I, II and III respectively) as compared to carcinogen control (100%) group.
The tumor yield and tumor burden were also considerably decreased as compared to carcinogen control groups. Tumor yield (mean ± S.D.) of 2 ± 1.4, 1 ± 1.3 and 1.7 ± 2.3 was obtained among groups I, II and III respectively as compared to control group (IV) giving tumor yield of 4.8 ± 2.8. Similarly, a tumor burden of 2.4 ± 1.1, 2 ± 1 and 3.3 ± 2.3 was observed in group I, II and III respectively as compared to control group with tumor burden 4.8 ± 2.8. Likewise tumor diameter was also found to be less in grapes treated groups i.e. 4.2 ± 1.9 in group I, 2.8 ± 1.1 in group II and 3.3 ± 0.9 in group III whereas tumor diameter of control group (IV) was calculated as 4.9 ± 1.4.
There were no major differences in body weights among the treatment groups during the study. Final body weights of mice (calculated in average ± S.D.) at the time of termination were 25.66 ± 2.86, 24.08 ± 1.77 and 25.01 ± 2.37 for group I, II and III respectively. However, there was decline from 21.83 ± 0.90 (Initial body weight) to 18.76 ± 3.57 in the final body weights of mice of carcinogen control group.
Above results can be correlated with the study of Zhao J. et al (1999) in which topical application of GSP at the doses of 0.5 and 1.5 mg resulted in quite high significant inhibition in TPA tumor promotion. A reduction in tumor incidence (35 and60%), tumor multiplicity (61 and 83% inhibition) and tumor volume (67 and 87% inhibition) at both the doses of 0.5 and 1.5 mg was observed respectively. Furthermore no noticeable difference was found in the average body weights of mice of GSP treated and control group from initiation till termination of experiment. Similar results were obtained in the study of Mittal A. et al. (2003) in which they evaluated the antiphotocarcinogenetic effect of dietary feeding of grape seeds proanthocyanidins (GSP) in SKH-1 hairless mice. They found that feeding of GSP at the dose of 0.2 and 0.5% prevented tumor incidence, tumor yield, tumor burden and tumor diameter amongst UVB alone, UVB + TPA and DMBA + UVB treated mice. Moreover there was no apparent body weight loss or extra weight gain among GSP fed mice as compared to non-GSP-fed mice i.e. control group which indicates non adversarial effect of GSP.
In a similar designed study by Bomser J.A. et al. (1999) it was shown that treatment of CD-1 mouse skin with 5, 10 and 20 mg of polyphenolic fraction of grape seeds (GSP) prior to TPA dosing effectively reduced the tumor incidence and tumor multiplicity. An eminent study was done by Meeran S.M. et al. (2009) in which they showed that dietary GSP inhibits TPA-promoted skin tumor development in DMBA-initiated mouse skin. Feeding mice a dietary concentration of 0.2 and 0.5% GSPs along with regular control diet AIN76A to C3H/HeN mice resulted in lower tumor incidence (20% and 35% lower), tumor burden (43% and 70% inhibition) and tumor size (32% and 70% lower) as compared to mice receiving control diet.
Another similar study by Hanausek M. et al. (2011) involved combinatorial effect of 1% dietary grape powder and 1 mg Resveratrol, a well known antioxidant found in grapes on DMBA initiated and promoted SENCAR mouse skin carcinogenesis. In their results they observed that grape powder and resveratrol in combination better inhibit tumor development as compared to the individual effects of 1 mg resveratrol or 1% Grape powder. They also monitored the body weights of mice weekly and found no change among the body weights of treated groups.
DMBA/Croton oil induced skin tumirogenesis was performed by Chaudhary G. et al (2011). They evaluated antitumor effect of Aloe vera on swiss albino mice. Notable reduction in tumor incidence, tumor yield and tumor burden was observed in mice groups treated topically with Aloe gel (group IV), orally with Aloe extract (group V) and topically with Aloe gel + orally with Aloe extract (group VI) as compared to DMBA/Croton oil treated control group.
In present study average body weights among mice of grape extracts treated mice was found to be increased as compared to control group which showed decline in average body weight of mice. The protection from body weight loss in grapes treated groups can be attributed to the presence of fibre, potassium, copper and a range of vitamins like vitamin K, vitamin B and minerals which benefits health. Resveratrol, a key nutrient present in grapes is considered to be the major source of its health promoting effects.
Methanolic Black grape extract showed highest reduction in tumor incidence, tumor yield, tumor burden and tumor diameter which was followed by methanolic green + black grape extract and methanolic green grape extracts respectively. The remarkable tumor inhibitory effect of methanolic black grape extract can be credited to its rich flavonoid content as described within present study during quantitative evaluation of phytochemicals. Flavonoids are considered as the main group of anticancer constituents in grape products. Moreover rich presence of phytonutrients likes anthocyanins, polyphenols and resveratrol helps in protecting free radical damage by ROS (Reactive Oxygen Species) thus counteracting tumor formation Zhou K. and Raffoul J.J. (2012).
Methanolic green + black grape extract also depicted increased and quite significant tumor inhibition. This antitumor activity can be attributed to the drug interaction between the two extracts. Methanolic green grape extract showed lesser tumor inhibition as compared to methanolic black grape extract, the drug-drug interaction resulted in a synergistic antitumor effect which enhanced the inhibition efficiently. The synergistic antitumor effect of grape seed proanthocyanidin and doxorubicin is well explained in the study of Zhang X.Y. et al. (2005). Present study indicates that carefully designed combinations of black grape phytochemical with green grape phytochemical(s) may be beneficial in cancer management.
However, DMBA/Croton oil control group showed 100% tumor incidence, 4.8 ± 2.8 tumor burden and tumor yield and 4.9 ± 1.4 mm tumor diameter. Previous studies suggests that reactive oxygen species (ROS) might play the key role in tumor initiation by increasing the metabolic activation and initiating effects of carcinogens Athar M. (2002) and induction of epidermal ornithine decarboxilase (ODC) Murakami A. et al. (2000).
Above data suggests that Black and Green grapes methanolic extracts possess potential to be an effective antitumor agent and the synergistic effect shown by these two drug combination can be a promising mode of attaining new ways to counteract dreadful diseases like skin cancer.
BIOCHEMICAL TESTS FOR OXIDATIVE STRESS
In present study potential of Green grape methanolic extract, Black grape methanolic extract and Green + Black grape methanolic extract to modulate oxidative stress in swiss albino mice was evaluated through Lipid Peroxidase test (LPO), Superoxide Dismutase (SOD) method and Reduced Glutathione (GSH) estimation.
LPO of Green Grape methanolic extract, Black grape methanolic extract and Green + Black Grape methanolic extracts was calculated to be 56.8 ± 2.5 nM MDA/gm wet tissue, 36.1 ± 2.8 nM MDA/gm wet tissue and 48.25 ± 4.2 nM MDA/gm wet tissue respectively. Similarly, SOD of Green Grape methanolic extract, Black grape methanolic extract and Green + Black Grape methanolic extracts was found to be 94.7 ± 12.1 U/gm wet tissue, 165.2 ± 12.4 U/gm wet tissue and 122.6 ± 10.1 U/gm wet tissue and GSH of Green Grape methanolic extract, Black grape methanolic extract and Green + Black Grape methanolic extracts was found to be 0.6 ± 0.01 nmol/gm wet tissue, 0.9 ± 0.04 nmol/gm wet tissue and 0.7 ± 0.03 nmol/gm wet tissue respectively. Whereas LPO, SOD and GSH of control group was calculated as 74.4 ± 3.9 nM MDA/gm wet tissue, 54.1 ± 6.5 U/gm wet tissue and 0.4 ± 0.02 nmol/gm respectively. This shows that grapes produced protection by decreasing the activity of lipid peroxidation and at the same time levels of GSH and SOD were increased significantly to near the normal levels.
Present study can be correlated with the study of BVS L. et al. (2014) observed that black grape extract helped in declining the high level of the lipid peroxide level in serum and elevating the levels of antioxidant enzymes SOD and GSH, thus minimized the noxious effects of cadmium. Similarly, Almajwal A.M. and Elsadek M.F. (2015) evaluated the hepatoprotective activity of red grape (Vitis vinifera L.) dried seeds. In their study it was found that administration of red grape dried seeds lowered the lipid peroxidation level while it increased the enzymatic levels of GSH and LPO. Similarly, Dani C. et al. (2008) compared organic purple grape juice and conventional purple grape juice (V. Labrusca) for their in vivo antioxidant activities. It was observed that organic grape juice was richer in antioxidants as compared to the conventional grape juice. Both the grape juices were able to alter the lipid peroxidation profile to low level and SOD to higher level. Vitis vinifera L. leaves were studied for their antioxidant and anti-hypercholesterolemic potential by Devi S. and Singh R. (2017) and it was observed that Vitis vinifera Methanolic extract and Vitis vinifera Aqueous extract appreciably elevated serum catalase level and serum reduced glutathione level. In the same way Dogan A. and Celik I. (2012) studied the hepatoprotective and antioxidant role of grape (Vitis vinifera L.) seeds against ethanol-induced oxidative stress in which the tissues of brain, spleen, kidney, heart and liver of treated rats were observed and it was concluded that administration of rats with alcohol and alcohol + grape seed extract resulted changes in the level of MDA content (LPO), SOD and GSH.
Giribabu N. et al. (2016) analysed Vitis vinifera L. seed methanolic extract (VVSME) to treat myocardial damage among diabetic patients. Heart homogenates were used to determine levels of oxidative stress and it was observed that VVSME consumption reduced the level of LPO while it improved the levels of SOD, Catalase and GPx. Pirinccioglu M. et al. (2012) and Rodrigues A.D. et al. (2013) examined Okuzgozu and purple grapes juices respectively for their activity against oxidative stress and found that grape juices were effective in lowering the lipid peroxidation and inhibiting the reduction in enzymatic antioxidant defences i.e. SOD and GSH. A polyherbal formulation PartySmart containing various fruits including Vitis vinifera L. was determined for its inhibitory effect against alcoholic liver disease by Gopumadhavan S. et al. (2008) and it was proved to reduce oxidative stress by lowering the lipid peroxidation level thus beneficial in the treatment of alcohol-induced liver damage.
Present study encompasses the defensive properties of Green Grape methanolic extract, Black Grape methanolic extract and Green + Black Grape methanolic extract against oxidative stress in swiss albino mice. From the results it was concluded that Black Grape methanolic extract is superior in preventing the damage caused due to oxidative stress followed by Green + Black Grape methanolic extract and Green Grape methanolic extract when compared to control group. In control group the level of lipid peroxidation showed a significant increase while the levels of GSH and SOD showed a significant decrease. The increased lipid peroxidation and decreased SOD and GSH level in control group might have resulted from an elevation of reactive oxygen species caused due to stress condition in the mice with DMBA/Croton oil intoxication.
Grapes (Both green and black) have strong antioxidant properties and thus can protect cells and tissues against free oxygen radicals. The protective effect of black grape methanolic extract might be due to the presence of flavonoids, catalase and phenolic compounds including anthocyanins and resveratrol in black grapes. All of these compound work together and impart with synergistic antioxidant effect, scavenging and eliminating free radicals Johnston et al. (2005).
Furthermore the mixture of Green Grape and Black Grape methanolic extract was found more effective in inhibiting oxidative stress as compared to Green Grape methanolic extract alone which implies that mixture of green and black grape methanolic extract synergistically enhance their in vivo antioxidant activities. The antioxidant properties of both black grapes and green grapes have been shown to suppress superoxide anion and lipid peroxidation and to delay the conjugation of dienes during lipid peroxidation Maffei-Facino et al. (1996). Both green and black grapes have been reported having amplified antioxidant properties. They help in avert from aging, recover skin and have huge amounts of fibre for digestion and vitamins to aid with immunity and strength.
So it can be concluded that when green and black grapes of varying potency were combined together they came out producing enhanced results as compared to the individual use of green grapes alone. Also, the positive interaction between both the grapes produced synergistic effect which can be pharmacodynamic synergism.