INTRODUCTION:

Wonderfully delicious, cherry fruit is packed with full of health-benefiting nutrients and unique antioxidants. Cherries are native to Eastern Europe and Asia Minor regions. Botanically, the fruit is a “drupe” (stone fruit), belonging to the broad Rosaceae family of small tree fruits in the genus, Prunus. Some of the common “drupe” family fruits are plums, peaches, apricots etc.

Although several species of cherries exist, two popular cultivars are wild or sweet-cherry, and sour or tart-cherry. While sweet cherries belong to the species of Prunus avium, tart variety belongs to that of Prunus cerasus. Cherries are drupe fruits with a central “stony-hard” seed surrounded by fleshy edible pulp measuring about 2 cm in diameter. Externally they covered by bright "shiny" red or purple, thin peel. The West Indian cherry, known as acerola (Malpighia emarginata) is native to West Indian islands and grown in Mexico, Texas regions in North America. Acerola belongs to tropical fruit-bearing shrub or small tree in the family Malpighiaceae and contains 2-3 tiny seeds. Acerola contains exceptionally high levels of vitamin-C and vitamin-A than North American and European cherries. Cherries are one of the very low calorie fruits. Nonetheless, they are rich source of phytonutrients, vitamins, and minerals. Both sweet as well as tart cherries are packed with numerous health benefiting compounds that are essential for wellness. Cherries are pigment rich fruits. These pigments, in fact, are polyphenolic flavonoid compounds known as anthocyanin glycosides. Anthocyanins are red, purple or blue pigments found in many fruits and vegetables, especially concentrated in their skin, known to have powerful anti-oxidant properties.

Fig: Cherry fruits

Scientific studies have shown that anthocyanins in the cherries are found to act like anti-inflammatory drugs by blocking the actions of enzymes cyclooxygenase-1 and 2. Thus, consumption of cherries may offer potential health effects against chronic painful episodes such as gout arthritis, fibromyalgia (painful muscle condition) and sports injuries. Research studies also suggest that anti-oxidant compounds in tart cherries can help the human body to fight against cancers, aging and neurological diseases, and pre-diabetes condition. Cherries compose of melatonin anti-oxidant. Melatonin can cross the blood-brain barrier easily and has soothing effects on the brain neurons, calming down nervous system irritability. It, thus, can help relieve neurosis, insomnia and headache problems [1-2].

Cherry texonomy[3]:

Cherries are members of the Rosaceae family, subfamily Prunoideae. They occupy the Cerasus subgenus within Prunus, being fairly distinct from their stone fruit relatives plums, apricots, peaches, and almonds. Prunus avium L. is the Sweet Cherry, and Prunus cerasus L. the Sour, Pie, or Tart Cherry.

Botanical description:

Plant: Sweet Cherry. Vigorous tree with strong apical control with an erect-pyramidal canopy shape, capable of reaching 50 ft. In cultivation, sweet cherries are maintained 12-15 ft in height. Leaves are relatively large (largest of cultivated Prunus), elliptic with mildly serrate margins, acute tips, petioled, and strongly veined.

Sour Cherry. Medium sized tree with a rounder, more spreading habit than the erect sweet cherry. Kept <15 ft in cultivation. Leaves elliptic with acute tips, mildly serrate margins, smaller than sweet cherry, with long petioles.

Flowers:

Sweet Cherry. White, with long pedicels, borne in racemose clusters of 2-5 flowers on short spurs with multiple buds at tips; the distal bud is vegetative and continues spur growth. Spurs are long-lived, producing for 10-12 years. Ovary position. is perigynous with a distinct hypanthium, characteristic of stone fruits. Sour Cherry. Individual flowers are the same as for sweet cherry. Sour cherry inflorescence buds usually produce 2-4 flowers, with long pedicels, as in sweet cherry. However, many are borne laterally on 1-yr wood, not exclusively on spurs as in sweets. Spurs are shorter-lived on sour than sweet, gradually declining in productivity over 3-5 years. Sour cherries are the latest blooming of the stone fruits.

Pollination:

Sweet Cherry. Pollination is absolutely essential for production, since sweet cherries are self-incompatible and need a high degree of fruit set (25-50%) for a commercial crop. In addition to self-incompatibility, there is a high degree of cross-incompatibility. Pollinizers are set every third tree in every third row, or a ratio of 8-9:1. Honey bees are the main pollinator.

Sour Cherry:

Sour cherries are self-fertile, and require no pollinizers.

Sweet Cherry:

A drupe; ½” to 1 1/4″, round or heart-shaped, glabrous, with long pedicel attached. The pit is generally smooth, and encloses a single seed. The skin color is generally deep red or purple (often referred to as “black”), yellow, or rarely white. Yellow fruit often have a red cheek. The flesh color varies from white to dark red. Fruit is borne on short spurs that arise from older wood. Sweet cherries require only about 2-3 months for fruit development. Thinning is unnecessary.

Pharmacological actions [4]:

Cherries are a nutritional powerhouse fruit with so many incredible health benefits. One cup of raw cherries has 87 calories, 22 grams of carbohydrates, 1 gram of protein and 3 grams of fiber. Enjoy them now while they are at their peak because their season is way too short. Read on for some of the great health benefits of eating cherries.

Ten Great Health Benefits of Eating Cherries:

1. Cherries, known as a “super-fruit”, are packed with antioxidants called anthocyanins which aid in the reduction of heart disease and cancer.

2. Cherries are one of the few food sources that contain melatonin, an antioxidant that helps regulate heart rhythms and the body’s sleep cycles.

3. Cherries are an excellent source of beta carotene (vitamin A). In fact they contain 19 times more beta carotene than blueberries and strawberries.

4. Cherries are rich in vitamins C, E, potassium, magnesium, iron, folate and fiber.

5. Cherries are referred to as “brain food”, aiding in brain health and in the prevention of memory loss.

6. Because cherries contain anthocyanins, they can reduce inflammation and symptoms of arthritis and gout.

7. Eating cherries reduces the risk of diabetes.

8. Cherries are a good source of fiber which is important for digestive health.

9. Cherries are a great snack or dessert choice important for weight-maintenance.

10. Because of their powerful anti-inflammatory benefits, cherries are said to reduce pain and joint soreness for runners and athletes after workouts.

MATERIALS AND METHOD:

Drugs and chemicals:

The standard drug doxorubicin purchased from Local Retail Pharmacy Shop and solvents and other chemicals used for the extraction and phytochemical screening were provided by Institutional Store and were of LR and AR grade.

Cell culture:

The human breast cancer cell line MCF-7 cell line was provided by National Centre for Cell Science (NCCS), Pune and was grown in Eagles Minimum Essential Medium (EMEM) which contained 10% fetal bovine serum (FBS). All cells were maintained at 37°C, 100% relative humidity, 5% CO2, 95% air and the culture medium was changed twice a week.

Apparatus:

Round bottom flask, water condenser, heating mantle, motor and pestle.

Methodology [5]:

Weigh 20 g of sweet cherry fruits paste (ripen can be mashed to prepare a paste) into a 250 ml round-bottomed flask. Add 50 ml of ethanol and 60 ml of dichloromethane. Heat the mixture under reflux for 5 min on stem-bath with frequent shaking. Filter the mixture under suction and transfer the filtrate to a separating funnel. Wash this mixture containing bioactive compounds with three portions of 150 ml each with sodium chloride solution. Dry the organic layer over anhydrous magnesium sulfate. Filter and evaporate most of the solvent in vacuum without heating and obtained ethanolic extract of sweet cherry (EEC) of Prunus avium. Same procedure was followed for the preparation of ethylacetoacetate extract of sweet cherry (EAAEC) of Prunus avium.

Phytochemical screening [6-8]:

Preliminary Phytochemical screening of ethanolic and ethyl acetoacetate extracts of sweet cherry of Prunus avium had shown the presence of various bioactive compounds such as carbohydrates, amino acids and peptides, phytosterols, carotenoids, and polyphenols etc.

Evaluation of anticancer activity of EEC and EAAEC of sweet cherry (Prunus avium) by MTT assay:

Principle [9]: The MTT assay is a colorimetric assay for assessing cell viability. NAD(P) H-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide to its insoluble formazan, which has a purple color. Other closely related tetrazolium dyes including XTT, MTS and the WSTs, are used in conjunction with the intermediate electron acceptor, 1-methoxy phenazine methosulfate (PMS).With WST-1, which is cell-impermeable, reduction occurs outside the cell via plasma membrane electron transport[36]. Tetrazolium dye assays can also be used to measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferation to quiescence) of potential medicinal agents and toxic materials. MTT assays are usually done in the dark since the MTT reagent is sensitive to light.

Screening of in vitro anticancer activity by MTT assay [10-12]

Cell culture:

The human breast cancer cell line MCF-7 was provided by National Centre for Cell Sciences, Pune, India and was grown in Eagles Minimum Essential Medium (EMEM) which contained 10% fetal bovine serum (FBS). All cells were maintained at 37°C, 100% relative humidity, 5% CO2, 95% air and the culture medium was changed twice a week.

Cell treatment:

The monolayer cells were detached and single cell suspensions were made using trypsin-ethylene diaminetetraacetic acid (EDTA). A hemocytometer was used to count the viable cells and the cell suspension was diluted with a medium containing 5% FBS in order to obtain final density of 1x10⁵ cells/ml. 96-well plates at plating density of 10,000 cells/well were seeded with one hundred microlitres per well of cell suspension and incubated for cell attachment at 37° C, 5% CO2, 95% air and 100% relative humidity. The cells were treated with serial concentrations of the test samples after 24 hr. Serial dilution method was used for preparing test extracts (EEC and EAAEC) of different concentrations. Cells were initially dissolved in dimethylsulfoxide (DMSO) and further diluted with serum free medium to obtain twice the desired final maximum test concentration. The required final extract concentrations of 10, 20, 30, 40 and 50 µg/ml were obtained by adding aliquots of 100 µl of the different extract dilutions to the appropriate wells already containing 100 µl of medium. After addition of the test extract the plates were incubated for an additional 48 hr at 37° C, 5% CO2, 95% air and 100% relative humidity. The medium without samples served as control and triplicate was maintained for all concentrations.

MTT assay:

After 48h of incubation, to each well 15µl of MTT (5 mg/ml) in phosphate buffered saline (PBS) was added and incubated at 37° C for 4h. The medium with MTT was flicked off and the formed formazan crystals were solubilized in 100 µl of DMSO. Using micro plate reader the absorbance was measured at 570 nm. The % cell inhibition was determined using the following formula:

% Cell Inhibition =

[100 - Abs (sample)/Abs (control)] x100.

RESULTS AND DISCUSSION:

The results for cell growth inhibition by the extracts such as EEC and EAAEC against MCF-7 cell lines for various concentrations is shown in table 1 and 2. As the concentration increases there is an increase in the cell growth inhibition and it was found that EEC with the highest 92.90% growth inhibition at 10 µg (IC₅₀ = 2.4 µg/ml) and EAAEC with the 92.49% growth inhibition at 10 µg (IC₅₀= 2.9 µg/ml). In the USNCI screening program a compound is generally considered to have in vitro anticancer activity, if the IC₅₀ value following incubation between 48 hrs and 72 hrs is less than 4 µg/ml or 10 µM. In the present study IC₅₀ values below 4 µg/ml were displayed by the various extracts of sweet cherry of Prunus avium. The IC₅₀ value of standard drug doxorubicin was found to be 1.3 µg/ml with 96.62 % growth inhibition at concentration 75 µg/ml.

Table 1: For percentage (%) of cell growth inhibition of ethanolic extract of cherry fruits (EEC) on MCF-7 Cell lines by MTT Assay

S. no.	Concentration of the Extracts	Absorbance of extracts	Inhibition of cell growth (%)
1	10 µg/ml	0.021	92.90
2	20 µg/ml	0.036	87.83
3	30 µg/ml	0.049	83.44
4	40 µg/ml	0.060	79.72
5	50 µg/ml	0.079	73.31
6	75 µg/ml of Dox. (Doxorubicin)	0.010	96.62
7	Control	0.296	0

Fig1: Percentage (%) of cell growth inhibition by EEC on human breast cancer MCF-7 cell line.

Table 2: For percentage (%) of cell growth inhibition of ethyl acetoacetate extract of cherry fruits (EAAEC) on MCF-7 Cell lines by MTT Assay:

S. no.	Concentration of the Extracts	Absorbance of extracts	Inhibition of cell growth (%)
1	10 µg/ml	0.022	92.49
2	20 µg/ml	0.038	87.16
3	30 µg/ml	0.052	82.43
4	40 µg/ml	0.066	77.70
5	50 µg/ml	0.081	72.63
6	75 µg/ml (Doxorubicin)	0.010	96.62
7	Control	0.296	0

Fig2: Percentage (%) of cell growth inhibition by EAAEC on human breast cancer MCF-7 Cell line.

CONCLUSION:

The results obtained from the present studies displayed that the Preliminary Phytochemical screening of ethanolic and ethyl acetoacetate extracts of sweet cherry of Prunus. avium had shown the presence of various bioactive compounds such as carbohydrates, amino acids and peptides, phytosterols, carotenoids, and polyphenols etc and results obtained from the in-vitro studies performed by MTT assay by using the human breast cancer MCF-7 cell line. MCF-7 cell lines displayed that the various extracts of sweet cherry (EEC and EAAEC) possessed a very good anticancer activity. From the present studied it had been concluded that EEC and EAAEC, all were exhibiting the potential capability to inhibit the cancer cell when compared with standard drug doxorubicin and the cell growth inhibition of EEC and EAAEC was found to be the highest 92.90% at 10 µg (IC₅₀ = 2.4 µg/ml) and 92.49% at 10 µg (IC₅₀= 2.9 µg/ml).

REFERENCES:

1. USDA National Nutrient Database.

2. Stanford School of Medicine Cancer information Page- Nutrition to Reduce Cancer Risk.

3. http://www.fruit-crops.com/cherry-prunus-avium-cerasus.

4. http://www.ingredientsinc.net/2011/08/top-10-health-benefits-of-cherries-a-true-superfruit.

5. Raj. K. Bansal ,Laboratory manual of organic chemistry , 5th revised edition , PP- 238-239.

6. P.C Dandiya, P.K. Sharma , Bio-chemistry and clinical pathology, second edition, PP- 17-18, 24, 47-48.

7. Dr. G. Devala Rao, A Manual of Practical Biochemistry, pp 17.

8. Jaswant Kaur, PV Chemistry of Natural Products, 2010 edition, PP-113-114, 116, 344-346, 381.

9. Berridge MV, Herst PM , and Tan AS. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnology Annual Review, 11: 127-152 (2005).

10. Sathish M, Tharani CB, Niraimathi V, Satheesh Kumar D, In-vitro cytotoxic activity on roots of Clerodendrum phlomidis against NIH 3T3 cell line and Hela cell line. Pharmacologyonline 2011; 3: 1112-1118.

11. Pranay Dogra, Study of Antibacterial and Anticancer Activity of Selected Trifoliate Plants. Biofrontiers 2009; 1(2): 48.

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Received on 29.04.2016 Modified on 28.05.2016

Research J. Pharmacology & Pharmacodynamics.2016; 8(2): 65-70

DOI: 10.5958/2321-5836.2016.00012.4: