Effect of Olea europaea L. and Juglans regia L. Extracts on Human Cancer Cell Line Viability with Studying of Hypoglycemic and Antiglycation Properties

There is an urgent and continuous need to discover new sources of medicinal plants to obtain useful compounds with health properties. So the purpose of this study was to investigate activity of Olea europaea L and Juglans regia leaves extract on Hela cell line viability, antidiabetic and Antiglycation. The aqueous extracts were obtained from leaves. Alloxan 180 mg /kg body was used to induce diabetes. Mice with blood glucose level of ≥200 mg/dl were considered as diabetic and were received 10 mg/kg of body weight of Extracts. For Antiglycation, SDS-PAGE (sodium dodecyl sulphate–polyacrylamide gel electrophoresis) were prepared for the appearance of high molecular weight products. Hela cell line were cultured in RPMI-1640 medium for MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay analysis. The results showed that the Original Research Article Dakah et al.; JPRI, 33(14): 1-7, 2021; Article no.JPRI.65731 2 mean of glucose levels decreasing in mice that treated with 10 mg/Kg extracts of O. europaea and J. regia. Also SDS-PAGE showed that extracts of J. regia were better than of O. europaea in inhibition of glycation induced protein cross-linking at all studied concentrations. MTT assay showed that the Cytotoxicity was increased with increasing the doses of extract and the cytotoxic effects of J.regia extract were higher than O.europaea at all concentrations. This study showed


INTRODUCTION
Plants and natural products have been shown to present interesting biological and pharmacological activities and are used as therapeutic agents. Medicinal plants play an important role in general health and treatment of diseases for thousands of years and still using in traditional medicine systems around the world [1].
Cancer is one of the main causes of human diseases, is cause by abnormal cells that division uncontrolled. It is evaluated that there will be approximately 20 million new cancer cases in the whole world by the end of the year 2025 [2], so there are needs for finding more effective and safe treatment options for cancers, that is which have promoted researchers all over the world to discover new anticancer agents; plants have been used in treating cancer historically because plants have ability to produce secondary metabolites; a lot of these natural products have pharmacological and biological activities and are used as chemotherapeutic agents [3].
Diabetes is also one of the most common chronic diseases in nearly all countries. Diabetes is ranked seventh among the diseases that are leading to cause death and it is ranked third when fatal complications are taken into the account, and the number of people suffering from the disease worldwide is increasing at an alarming rate with a projected 366 million people likely to be diabetic by the year 2030 compared to an estimated 171 million sufferers in 2000 [4]. Protein glycation plays a key role in the development of chronic complications associated with diabetes such as neuropathy, nephropathy retinopathy and atherosclerosis [5][6], Protein glycation is initiated as a non-enzymatic reaction between reducing sugars and proteins followed by a series of reactions leading to formation of advanced glycation end products (AGE).
Alloxan is one of the chemical compounds used for the induction of diabetes [7][8]. The diabetogenic dose of alloxan varies considerably amongst species, age and metabolic state of the animal. Alloxan induced diabetes mechanism: Alloxan is reduced to dialuric acid and reoxidized to alloxan producing alloxan radicals and reactive oxygen species (ROS) which undergo dismutation (by superoxide dismutase, SOD) to form hydrogen peroxide (H 2 O 2 ). Hydroxyl radicals (*OH) may also be formed by side reactions. These *OH cause β-cell DNA fragmentation, leading to apoptosis [9].
Several studies confirm the potential therapeutic efficiency of some medicinal plants in treatment of diabetic patients and prevent glycation especially polyphenols and other natural antioxidants, so the daily intake of natural products can play a beneficial role in preventing the diseases. Many traditional treatments have been recommended in the alternative system of medicine for treatment of diabetes mellitus. Olea europaea L. with phenolic contents [10] and Juglans regia L. with flavonoids [11] are two of the medicinal plants that are used as a treatment for diabetes. Plants have historically been used in treating cancer and are recognized for their ability to produce secondary metabolites. There is an urgent and continuous need to discover new sources of medicinal plants to obtain useful compounds with health properties [12]. So the purpose of this study was to investigate antidiabetic effects of O. europaea L and J. regia leaves extract in Alloxan-diabetes induced mice, and to study Antiglycation effects against induced protein cross-linking and to investigate activity on Hela cancer cells viability.

Collection of Sample
The J. regia L. and O. europaea L. leaves were collected from Amman, Jordan. Mice and Hela cell line were obtained from Biotechnology and genetic engineering department, Philadelphia University.

Extraction Procedure
The leaves were washed with distilled water and placed in oven at 37°C to dry. Then, the leaves were completely pulverized. 200 g was soaked in 1000 ml distilled water and placed on incubator with shaking for 24 hours at 37°C. The solution was filtered through a filter paper and dried in the oven at 39°C [13].

Experimental Animal
Adult and healthy mice (Mus musculus ) of 30-35 g were used.

Diabetic Induction
Alloxan was used to induce diabetes. Mice (33 g) were injected with subcutaneous injection 180 mg /kg body (5,94 mg/ 33 g) of freshly prepared of alloxanin [14]. Diabetes mellitus was confirmed by testing blood glucose after 24 hours, and mice with blood glucose level of ≥200 mg/dl were considered as diabetic.

Experimental Design
The total of 40 mice (n= 10) were grouped randomly into 4 groups as follows: Group I:

Detection of Glycation Induced Protein cross-linking
Working solutions were prepared as described by Perera

SDS-Page
SDS-polyacrylamide gels (12%) were prepared according to Laemmli [16] for the appearance of high molecular weight products. Aliquots from the incubation mixtures were heated with the SDS sample buffer at 95°C for 3 min, before loading to the gel. Molecular weight markers (10-225 kDa) were used to assess the approximate size of the high molecular weight products. After separation at pH 8.6, protein bands were visualized by staining with Coomassie blue.

Culture of Hela Cells
Hela cells were cultured in RPMI-1640 medium with 10% FBS and 1% antibiotics, Maintained at 37°C and 5% CO2 atmosphere, Cells were subcultured every 2-4 days with trypsin/EDTA.

MTT Assay for Cytotoxicity
Cells were seeded in a 96-well flat-bottom microtiter plate at a density of 1 × 104 cells/well and allowed to adhere for 24 hours at 37°C in a CO2 incubator. After 24 hours of incubation, culture medium was replaced with a fresh medium. Cells were then treated with various concentrations (6.25, 12.5, 25, 50 and 100 µL) of plant extracts for 24 hours at 37°C in a CO2 incubator. After 24 hours of incubation, culture medium was replaced with a fresh medium. Subsequently, 10 μL of MTT working solution (5 mg/mL in phosphate buffer solution) was added to each well and the plate was incubated for 4 hours at 37°C in a CO2 incubator. The medium was then aspirated, and the formed formazan crystals were solubilized by adding 50 μL of DMSO per well for 30 min at 37°C in a CO2 incubator. Finally, the intensity of the dissolved formazan crystals (purple color) was quantified using the ELISA plate reader at 540 nm. And the viability was calculated from following formula: Viability% = (Test OD -blank OD)/ (Control OD -blank OD) x 100

Antidiabetic Effects
The results showed that the mean of glucose levels of normal control mice were about 111.5 ± 0.05 mg/dL, while the diabetic control mice that received Alloxan were significantly higher than control about 440 ± 0.01 mg/dL. Mice that treated with 10 mg/Kg of O. europaea and J. regia extracts showed decreasing in the glucose levels to become (197 ± 0.07 mg/dl and 181.5 ± 0.08 mg/dl) respectively after 7 days of treating diabetic mice, Table 1 and Fig. 1. The antidiabetic effect of an alcohol extract of Olea europaea L. leaves was studied, where the streptozotocin-induced diabetic rats received oral administration of the leaves extract for 14 days and showed significantly decreased the serum glucose [17]. In another study, blood sugar decreased meaningfully when alloxan-induced diabetic rats were treated with leaves extracts of J. regia (200 mg/kg) [18]. The antidiabetic effect is due to the presence of phenolic and flavonoids compounds in the extracts, the flavonoids supports the regulation of carbohydrate digestion, insulin secretion and glucose uptake [19]. A study on 200,000 women and men estimated the combination between dietary intake of flavonoids and type 2 diabetes lowers the risk of diabetes [20].

Detection of Glycation Induced Protein Cross-Linking
SDS-PAGE of aliquots were collected and compared after 20 days of incubation in the presence of plant extracts. SDS-PAGE showed that extracts of J. regia were better than extracts of O. europaea in inhibition of glycation induced protein cross-linking at concentrations (5, 10, 15 and 20 mg/ml) (Fig. 2). So, J. regia has high antiglycation properties and agree with Ahmad and his colleagues who supposed that J. regia extract could have therapeutic uses in inhibit chronic diabetic complications and slowing aging [21]. Extracts of J. regia at all used concentrations (5, 10, 15 and 20 mg/ml) prevented formation trimer (~36 KDa) and tetramer (~48 KDa) products, while high molecular weight products trimer and tetramer of lysozyme appeared when lysozyme was incubated in the presence of O. europaea extract at concentrations (5, 10, 15 and 20 mg/ml). Lysozyme was used as a model protein in many studies to display glycation associated protein cross-linking [22]. This current study gave an idea about the approximate inhibition to the cross-link formation using extracts by visual observation. Many antidiabetic plants and spices showed cross-link inhibitory effects, extracts of Pterocarpus marsupium and Coriandrum sativum have shown inhibitory effects on glycation induced protein cross-linking and Syzygium aromaticum showed complete inhibition on the formation of cross-linking with 25 μg/ml extracts [23]. In our study, results with fructose clearly demonstrated that the extent of cross-linking was good because its effects occur at a more manageable rate, and Perera and Ranasinghe proposed that fructose is better than glucose and ribose, in detecting glycation induced cross-link formation [15].

MTT Assay
The results obtained from this study using MTT assay (Viability%) showed the Cytotoxicity of J.regia and O.europaea extracts on death of Hela cells compared to control group. The inhibitory effect of plant extract was dependent on concentration of extract and the species of plants, in Table 2 and Fig. 3 the Cytotoxicity was increased with increasing the doses of extract and the cytotoxic effects of J.regia extract were higher than O.europaea at all studied concentrations. The highest Cytotoxicity was observed at 100 µL after 24 hours at 37°C in a CO2 incubator. Also, the percentage of cell survival was lower at 6.25 µL. These anti-cancer properties in O.europaea are thought to presence phenolic compounds, especially oleuropein and hydroxytyrosol, oleuropein and hydroxytyrosol were studied for its effects on growth in MCF-7 human breast cancer cells using MTT assay, these compounds decreased cell viability and induced cell apoptosis in MCF-7 cells [24]. Oleuropein present in O. europaea leaves gave it very important scientific value due to its several biological properties including anticancer, Ruzzolini and his colleagues found that Oleuropein at a dose of 500 µM was able to stimulate apoptosis, while at dose of 250 µM was non-toxic [25]. Salimi et al. [3] assayed Cytotoxicity effects of various J. regia leaf extracts in Human oral squamous carcinoma (BHY), colon adenocarcinoma (HT-29) and breast adenocarcinoma (MCF7) cell lines with MTT assay. Their results indicated that MCF7 and BHY cells were best inhibited by total extracts after 48-h treatment, while HT-29 cells were not inhibited by them [3].

CONCLUSION
We have demonstrated that both of J. regia and O.europaea aqueous leaves extracts have properties as antidiabetic, antiglycation and anticancer, but J. regia was more effectively.

CONSENT
As per international standard or university standard, patient's written consent has been collected and preserved by the author(s).

ETHICAL APPROVAL
Animal Ethic committee approval has been collected and preserved by the author(s).