In vitro evaluation of antioxidant, antineurodegenerative and antidiabetic activities of Ocimum basilicum L., Laurus nobilis L. leaves and Citrus reticulata Blanco peel extracts

Ocimum basilicum (sweet basil) and Laurus nobilis (bay leaves or laurel) have been used in traditional medicine for centuries, and also extensively employed as spices for adding aroma and flavor to various food products. Citrus reticulata (mandarin) is mainly used in food industry for juice production, while its peel as main byproduct contains high concentration of valuable substances. The samples were collected in Lastva Grbaljska (Montenegrin coast) and purchased from the market. Since the oxidative stress results in development of numerous diseases, among them neurodegeneration and diabetes, the antioxidant activity, antineurodegenerative and antidiabetic activities were analyzed, aiming to compare potential of plants cultivated under natural conditions and commercially purchased from the market, as well as to compare the effect of different solvents applied in the extraction process. Water, methanol and acetone extracts of leaves and peel were tested by DPPH and total reducing power (TRP) methods for determination of antioxidant activity, and by acetylcholinesterase (AChE) and αglucosidase inhibition assays for analyzing the other activities. Total phenolic (TPC) and flavonoid (TFC) contents were also determined. The acetonic extract of L. nobilis from Lastva showed the highest TPC, DPPH, TRP, and α-glucosidase inhibition, while water extract of commercial L. nobilis exhibited the highest AChE inhibition. The leaves of L. nobilis are demonstrated to be promising antioxidant, antineurodegenerative and antidiabetic agent.


INTRODUCTION
Free radicals are highly reactive species having unpaired electrons in their outermost shell leading to oxidative stress, which causes tissue damage and results in large number of diseases. Oxidative stress plays a major role in progression of neuro-logical diseases, such as Alzheimer's disease (AD) and also in diabetes mellitus (DM). When disbalance of the production of reactive oxygen species (ROS) and cellular antioxidant defenses appears, proteins and nuclear acids damages occur, having destructive effects in AD and DM (Reddy et al., 2009). Numerous molecular, clinical, epidemiological, etc. data supports a pathophysiological link between AD and DM (Ahmad et al., 2017). Cognitive decline related to DM is characterized by mild to moderate impairment, and an increased risk of developing AD and other forms of dementia (Toth, 2014). Considering the fact that AD and DM reach epidemic proportions, different approaches for their prevention and treatment are present among scientists worldwide. Natural products are extensively studied in the last decades for suppressing ROS production, and might be promising in AD and DM therapy. Antioxidants of natural or synthetic origin neutralize the effects of reactive oxygen species and thus help in preventing diseases. There is a growing interest both in various industrial sectors and among people worldwide towards the use of natural compounds obtained from plants in cosmetic, food and pharmaceutical industries which could represent alternatives to synthetic chemicals, because natural compounds have lesser environmental and human health impacts. Natural antioxidants can be taken up through diet as they are present in fruits, vegetables and spices which contain a large amount of flavonoids and antioxidant supplements that contribute to protection against different types of health problems (Hamid et al., 2010). The beneficial effects of medicinal plants in AD and DM have been confirmed in numerous studies (Bazzari and Bazzari, 2018;Nasri et al., 2015). Several species belonging to the family Lamiaceae are of high socio-economic value, having therapeutical, culinary, horticultural, ornamental or industrial applications due to the high content of bioactive phenolic compounds (Trivellini et al., 2016). The Lauraceae family comprises species which contain considerable concentrations of essential oils, including genera of high commercial value, such as cinnamomum, laurus and persea. Different kinds of Citrus fruits are used in food industry for fresh juice production, with the peel as the main waste fraction, which had been widely studied because of high content of numerous biologically active compounds including natural antioxidants such as phenolic acids and flavonoids (Xu et al., 2007). Ocimum basilicum L. (Lamiaceae), commonly known as sweet basil, is a perennial herb, native to Asia, Africa, South America, and the Mediterranean but widely cultivated in many countries (Grayer et al., 1996). This important medicinal plant and culinary herb has been used traditionally for treatment of anxiety, diabetes, cardiovascular diseases, headaches, nerve pain, cough, cold, fevers, headaches and migraines, insect bites, menstrual cramps, sinusitis, as an anticonvulsant, antiinflammatory and antithrombotic agent, and in a variety of digestive and neurodegenerative disorders (Purushothaman et al., 2018). Laurus nobilis L. (Laureaceae), commonly known as bay leaves or laurel, is a native plant from the Mediterranean region and is cultivated mainly in Europe and USA as an ornamental plant (Garg et al., 1992). It is widely used as a spicy fragrance and flavor in traditional meat dishes, stews and rice (Dias et al., 2014). Its leaves and extracts are used to suppress high blood sugar, fungal and bacterial infections, to treat eructation, flatulence and gastrointestinal problems and also exhibits anti-inflammatory, anticonvulsive, antiepileptic, cytotoxic, antioxidant and neuroprotective properties (Barla et al., 2007;Dias et al., 2014;Maajida Aafreen M et al., 2019;Pacifico et al., 2014). China is the most important centre of origin for the species of the genus Citrus (Zhang et al., 2014). Citrus species (Rutaceae) produce widely used fruits of high nutritional and industrial value, rich in flavonoid derivatives and polymethoxylated flavonoids, with different distribution in fruit parts (Nogata et al., 2006). Dried peel of Citrus reticulata Blanco is recorded in the Chinese Pharmacopeia as chenpi used for activation of vital energy and circulation, elimination of phlegm, disperse physical stagnation, etc., being good source of phenolics, especially of flavanone glycosides (Tumbas et al., 2010). The mentioned substances may play a role in the prevention of diabetes, cardiovascular and other diseases (Zhang et al., 2014). The main goal of this research was to compare bioactivities (antioxidant, antineurodegenerative and antidiabetic) of extracts of O. basilicum and L. nobilis leaves and C. reticulata peel obtained from plants cultivated under natural conditions in the Montenegro coastal region and commercially purchased from the market, and additionally to compare the influence of the different solvents applied in the extraction process.

Plant material
Plants were cultivated in Lastva Grbaljska, near Budva (Montenegro). Leaves of Ocimum basilicum and Laurus nobilis were collected in August 2018, while Citrus reticulata fruits were collected in November 2017. Plant material was dried and kept in shade at room temperature for further processing. The voucher specimens of L. nobilis and O. basilicum were deposited in the Herbarium of the Institute of Botany and Botanical Garden "Jevremovac", University of Belgrade, Faculty of Biology (BEOU; voucher No. 17510, and 17511). The commercially purchased basil and bay leaves are products of Premia (Serbia), while mandarins were purchased from the local market.

Preparation of extracts
Grinded plant material (5 g of O. basilicum and L. nobilis leaves and 10 g of C. reticulata peel) was extracted during 24 h at room temperature (5% w/v and 10% w/v, respectively) using acetone, methanol and hot water. The mixture was exposed to ultrasound 1 h before and after 24 h to improve the extraction process. Subsequently, extracts were filtered through filter paper (Whatman No.1) and evaporated under reduced pressure (Buchi rotavapor R-114). The obtained crude extracts were stored in a refrigerator at +4°C for further experiments. The yield of the extracts was calculated using the following equation: where m represents the mass of dry extract, while M is the mass of the dry plant extract used for the extraction.

Determination of total phenolic and total flavonoid contents
Total phenolic content (TPC) of the plant extracts was performed applying the spectrophotometric procedure of Singleton and Rossi (1965). The reaction mixture was prepared by mixing 100 µL of extract at the concentration of 0.5 mg/mL with 500 µL of 10% Folin & Ciocalteu's reagent dissolved in water. After 6 min, 400 µL of 7.5% sodium carbonate was added. Blank contained distilled water instead of extracts. The absorbance was measured at 740 nm after 2 h incubation at room temperature, using Jenway 7315 UV/Visible spectrophotometer. The same procedure was repeated for the standard solution of gallic acid (GA). The phenolic content of the samples was calculated from the standard curve and expressed as mg GA equivalents per gram of dry extract, presented as mean ± standard deviation. Total flavonoid content (TFC) of the samples was measured spectrophotometrically using Jenway 7315 UV/Visible spectrophotometer, according to the procedure of Park et al. (1997). The reaction mixture was prepared by mixing 0.5 mL of extract at the concentration of 0.5 mg/mL with 2.05 mL 80% ethanol, 0.05 mL 10% (Al(NO 3 ) 3 × 9H 2 O), and 0.05 mL 1 M CH 3 COOK. Blank contained 96% ethanol instead of extract. After 40 min of incubation at room temperature, the absorbance was measured at 415 nm. The same procedure has been repeated for the 96% ethanol solution of quercetin (Q) in order to construct the calibration curve. The content of flavonoids in the samples was expressed as mg Q equivalents per gram of dry extract, as mean ± standard deviation.

DPPH assay
DPPH free radical scavenging method (Blois, 1958) was used for the determination of antioxidant activity, with slight modification. In test tubes was added 100 µL of extract at the concentration of 0.5 mg/mL and 900 µL methanolic solution of DPPH (40 µg/mL). Methanol was used as blank, methanol with DPPH solution was used as negative control, while BHA, BHT and ascorbic acid were used as positive controls (standards). The absorbance was measured at 517 nm after 30 min in the dark at room temperature, using Jenway 7315 UV/Visible spectrophotometer. The decrease in absorption of DPPH was calculated as follows: where A c represents the absorbance of the negative control, while the absorbance of the test samples is labeled with A s . The results are presented as percentage of DPPH inhibition ± standard deviation.

Total reducing power assay
The total reducing power (TRP) was determined according to the method of Tusevski et al. (2014), with slight modifications. In the reaction mixture was added 100 µL of sample at the concentration of 0.5 mg/mL, 200 µL phosphate buffer (0.2 M, pH 6.6) and 200 µL 1% potassium ferricyanide. After the incubation (20 minutes at 50°C), 200 µL 10% trichloracetic acid, 200 µL distilled water and 40 µL 0.1% iron (III) chloride were added. Blank was prepared in the same manner, with the exception of adding 100 µL of adequate solvent instead of sample. The absorbance was measured at 700 nm after the 10 minute incubation at room temperature, using Jenway 7315 UV/Visible spectrophotometer. The total reduction power of the samples was presented as µmol of ascorbic acid equivalents (AAE) per gram of dry extract (µmol AAE/g dry extract).

Antineurodegenerative activity
Acethylcholinesterase (AChE) inhibitory activity assay was performed according to the procedure of Ellman et al. (1961), using 96-well plates. The reaction mixture (S) was prepared by adding 140 µL of sodium-phosphate buffer (0.1 M, pH 7), 20 µL DTNB solution, 20 µL sample (at the concentration of 0.5 mg/mL) and 20 µL AChE solution (5 U/mL) in Tris-HCl buffer (20 mM, pH 7.5). The mixture without the sample was used as a control (C, sodium-phosphate buffer instead of sample), the mixture without the enzyme was used as blank for the extracts (B), while galanthamin was used as positive control (at the concentration of 0.1 mg/mL). After 15 minutes at 25°C , the reaction was initiated by adding 10 µL of acetylcholine iodide. Absorbance was measured at 412 nm, using Multiscan Sky Thermo Scientific, Finland. The percentage of inhibition was calculated by using the following equation:

Antidiabetic activity
Determination of α-glucosidase inhibitory activity was performed according to the method of Wan et al. (2013), with slight modifications. Briefly, the same volumes (20 µL) of phosphate buffer (0.1 M, pH 6.9), α-glucosidase (0.5 U/mL) and samples (S) at the concentration of 0.5 mg/mL were preincubated for 5 min at 37°C. After that, 20 µL of pNPG (15 mg/10 mL) as substrate was added to the mixture and the incubation continued for another 20 min at 37°C. At the end, the reaction was stopped by adding 80 µL of 0.2 M sodium carbonate and the absorbance was measured at 405 nm using Multiscan Sky Thermo Scientific, Finland. The control (C) contained buffer solution instead of sample, while blank (B) contained buffer solution instead of enzyme. Acarbose at the concentration of 0.1 mg/mL was used as a positive control. The percentage of inhibition of α-glucosidase was calculated according to the following equation:

Statistical analysis
All experimental measurements were carried out in triplicate and the results are expressed as the average of three measurements ± standard deviation. To assess the potential effects of the solvent type, the results were subjected to one-way ANOVA, followed by Tukey's post-hoc test (the results were considered statistically significant at P<0.05 level). Differences between localities for all observed parameters were estimated through Student's t-test (P<0.05). Pearson's correlation coefficients (r) were calculated among the investigated activities of extracts and phytochemical contents and presented according to Taylor (1990), where r<0.35, 0.36<r<0.67 and 0.68<r<1 were considered weak, moderate and strong correlation, respectively. All statistical analyses were done using the software package STATISTICA v.10.0.

Yields, total phenolic and flavonoid contents of extracts
The yields of extracts varied depending on the plant species, origin of plant material and applied solvent ( Table 1). The highest extraction yields for O. basilicum leaves were achieved when water was used as an extraction solvent (10.08 and 10.54%). Bomma et al. (2018) analyzed six Ocimum species and obtained yield for methanolic extracts of O. basilicum ranging from 0.98 to 1.44%, while methanolic extracts in our research yielded 4.62 and 6.74%. In the case of bay leaves, the yield of acetonic extract of commercial sample (31.06%) was noticeable higher comparing to other samples. On the contrary, methanolic extracts of Moroccan bay leaves showed the highest yield (22%) comparing to other applied solvents (Taroq et al., 2018). Mandarin peel showed the highest yield in methanolic extracts of both commercial and naturally cultivated samples (23.07 and 23.77%) ( Table 1). Senol et al. (2016) in the study of the ethanolic extracts of peels of C. reticulata cultivars obtained yields from 11.39 to 42.5%. Safdar et al.
(2017) tested mandarin peels with different extraction methods and extraction solvents, and achieved the highest yield  Kivrak et al. (2017) have found that ethanolic extract of L. nobilis possess higher TFC than water extract, which is similar to our results, with noticeable higher TFC in methanolic extracts. Likewise, the ethanolic extract of bay leaves from Morocco had the highest TFC (Taroq et al., 2018). Zhang et al. (2014) notified that TFC varied from 6.28 to 20.66 mg RE/g for methanolic extracts of peels of Chinese mandarin genotypes. Zhang et al. (2018) have found that TFC for Chinese C. reticulata methanolic extracts ranged from 23.29 to 56.52 mg RE/g, which was evidently higher compared to our results (6.34-8.05 mg QE/g). Generally, literature data demonstrate that the TPC and TFC vary depending on the locality, season, applied solvent and the extraction procedure (Javanmardi et al., 2003;Kivrak et al., 2017;Muñiz-Márquez et al., 2014;Safdar et al., 2017). In our study (Table 1) the statistical analysis showed that the sample origin had a significant influence on both TPC and TFC. The used solvent had statistically significant impact to TPC and TFC in majority of tested extracts.

Antioxidant activity
The antioxidant activity was evaluated applying DPPH assay which has been widely used for the determination of free radical scavenging capacity of samples and total reducing power assay (TRP) which evaluates potential of sample to reduce potassium ferricyanide (Fe 3+ ) to form potassium ferrocyanide (Fe 2+ ). The results for antioxidant activity are presented in the Table 2. Acetonic extracts of leaves of L. nobilis showed the highest antioxidant activity in both assays. Likewise, water extracts of O. basilicum leaves exhibited high antioxidant potential. The influence of the solvent was statistically significant in both applied assays. The origin of the plant material was statistically significant in TRP assay for all species, while in DPPH test only in the case of L. nobilis. Kaurinović et al. (2011) have obtained the best results in DPPH assay among the investigated extracts for water one of O. basilicum, which is in accordance with our results. Zakaria et al. (2008) showed that methanolic extracts of O. basilicum leaves inhibited 45.35% of DPPH radical at the concentration of 1 mg/mL, while in our study, commercial O. basilicum methanolic extract achieved 36.14%, and cultivated O. basilicum methanolic extract 33.92% at the concentration of 0.5 mg/mL. In the research of Ahmed et al. (2019) ethanol extracts of basil from different locations exhibited stronger radical scavenging activity than that of synthetic antioxidant BHT, while in our study standards were significantly stronger than extracts in applied concentrations. Antioxidant activity study of Kivrak et al. (2017) showed that water extract of L. nobilis had rather lower activity in all the applied assays compared to ethanolic extracts. Their findings are in accordance with our results for both used methods. Methanolic extract of L. nobilis possessed effective reducing power in the study of Pacifico et al. (2014), while in our research it was less potent comparing to acetonic extract. Water extract of Moroccan bay leaves exhibited the highest DPPH radical scavenging activity comparing to other tested extracts, which was probably due to its high content of phenols (Taroq et al., 2018). Our results showed higher antioxidant activity of acetonic extracts than methanolic extracts. Dias et al. (2014) compared leaves of wild and cultivated L. nobilis plants and found that infusions of both samples revealed higher antioxidant activity than methanolic extracts, while in our study the methanolic extracts exhibited higher DPPH activity compared to water extracts. Water extract of Portuguese L. nobilis showed DPPH activity of 61% (Ferreira et al., 2006), similar as it was obtained in our research for commercial sample (59.89%) at the same concentration. Antioxidant activity of mandarin peels was studied by Xu et al. (2008) and they concluded that hot water extraction was effective in extracting of antioxidant compounds in Citrus peels, which was also the case in our study, with the highest antioxidant capacity obtained for the water extract of commercial sample (16.02%). Chen et al. (2011) have obtained considerably better results for ethanolic C. reticulata peel extract compared to our results. Zahoor et al. (2016) and Safdar et al. (2017) demonstrated high efficiency of methanolic extracts of C. reticulata from Pakistan, while methanolic extracts in our research had the lowest antioxidant activity. Zhang et al. (2014) reported that the DPPH values of the methanolic extracts of wild mandarins peel varied from 29.04 to 50.46 µmol Trolox equivalents/g. The FRAP values of the 14 wild mandarins, obtained by Fe 3+ reduction assay similar to TRP used in this research, ranged from 26.50 to 46.98 µmol TE/g DW in the study of Zhang et al. (2014).

Antineurodegenerative activity
The analysis of potential antineurodgenerative activity of various extracts of leaves and mandarin peel was conducted and the results of inhibition of acetycholinesterase, which is therapeutic target for AD, are presented in Table 3. The highest AChE inhibition was achieved by water extract of commercial L. nobilis, followed by acetonic and methanolic extracts. Among tested samples of O. basilicum, methanolic extracts showed higher activity compared to other extracts, while the mandarin peel was the most effective in the water extracts (Table 3). The AChE inhibition was significantly affected by both plant origin and the extraction solvent. Pharmacological studies on O. basilicum have demonstrated potent antioxidant activities with some reports of neuroprotective actions (Manali et al., 2018). Basil essential oils were much more investigated for AChE inhibition than its extracts. The results of Sarahroodi et al. (2012) indicated that 80% ethanolic extract of O. basilicum significantly increased memory retention and retrieval of mice. Among ethanolic and water extracts of ten Portuguese plants analyzed by Ferreira et al. (2006), L. nobilis showed AChE inhibitory activity of 19.9% for water extract, but much higher for ethanolic extract (48.4%) using concentrations of 0.5 mg/mL. In our research at the same concentration, the commercial L. nobilis displayed higher activity of water extract (90.72%) comparing to methanolic and acetonic extracts. Senol et al. (2016) in their study of ethanolic extracts of peels of 17 Turkish Citrus cultivars notified that extracts at the concentration of 0.5 mg/mL did not affect AChE activity, while in our study, at the same concentration, the mandarin peels showed AChE inhibition ranging from 8.34 to 46.12%. El-Khadragy et al. (2014) tested the methanolic extract of mandarin peel on memory impairment in rats produced by scopolamine, and concluded that administration of mandarin peel extract may be of value for dementia exhibiting elevated brain oxidative status.

Antidiabetic activity
Diabetes mellitus has been proved to be linked to cognitive decline and neurodegeneration in general. Alpha glucosidase inhibitors delay carbohydrate absorption in the gastrointestinal tract, control postprandial hyperglycaemia and reduce the risk of cardiovascular and neurological complications in the development of the disease (Kalra, 2014). The results of α-glucosidase inhibition are presented in the Table 3. The best results were obtained for water extract of O. basilicum and acetonic and methanolic extracts for L. nobilis originated from Lastva. The inhibition was not detected for the C. reticulata peels. Origin of the plant material and applied solvent significantly affected the α-glucosidase inhibition in the case of O. basilicum and L. nobilis (Table 3). Malapermal et al. (2017) showed that O. basilicum ethanolic extracts (70% and 60% ethanol) had higher antidiabetic activity than aqueous extract, which is similar to our results in the case of commercial sample. However, aqueous extract of O. basilicum from Lastva had the third best activity of all of the tested samples, which was far higher than the activity of the methanolic extract. The majority of data on the α-glucosidase inhibitory activity of L. nobilis is regarding to the essential oil, while the information on extracts is scarce. However, Kazeem et al. (2016) tested acetone extracts of bay leaves which displayed high antiglycation and antioxidant potential. Indrianingsih et al. (2015) have found that L. nobilis methanolic extract at the concentration of 0.2 mg/mL inhibited 47.26% α-glucosidase, which is in agreement with the results obtained in this study for the cultivated L. nobilis. Fayek et al. (2017) investigated the antidiabetic potency of different Citrus peel extracts and showed that mandarin peels decreased the glucose level in rats. Oboh and Ademosun (2011) found that α-glucosidase inhibitory effect of the Shaddock (Citrus maxima) peel acetonic extract achieved 89.05% at the tested concentration of 0.32 mg/mL. The enzymatic activity of α-glucosidase was tested for Citrus limetta peel extract and was found that at the lowest tested concentration (1.125 mg/mL) 5.2% enzyme was inhibited (Padilla-Camberos et al., 2014). It is considered that dietary α-glucosidase natural inhibitors are safe to control hyperglycemia, and medicinal plants could be useful remedies in treatment of diabetes and other health disorders (El-Beshbishy and Bahashwan, 2012).

Correlation between antioxidant activities, enzyme inhibition and total phenolic and flavonoid content
Pearson's correlation coefficients were calculated between total phenolic and flavonoid contents of tested extracts and their antioxidant activities and α-glucosidase (AGLU) and AChE inhibition (Table 4). Antioxidant activity was more strongly correlated to total phenolic than total flavonoid content which is in accordance to Wojdylo et al. (2007). Antioxidant potential of the plant material usually correlates well with the phenolic content, which was also the case in the study of different extracts from fresh, frozen and lyophilized basil leaves (Złotek et al., 2016), where the correlation between phenolic content and antioxidant activity was positive and varied regarding to the used material. TPC displayed moderate correlation to AGLU and AChE inhibition activities. Polyphenols seem to be mostly involved in antioxidant activity and enzyme inhibition thereby possessing therapeutic potential for AD and DM. Antioxidant assays were strongly correlated to AChE inhibition, and moderately to α-glucosidase inhibition, while the enzyme inhibition tests were moderately correlated.  Taylor (1990), where A (r<0.35), B (0.36<r<0.67) and C (0.68<r<1) were considered weak, moderate and strong correlation, respectively.

CONCLUSION
Among tested samples, O. basilicum and L. nobilis cultivated in Montenegro had higher level of antioxidant activity in both assays and also of α-glucosidase inhibition, while water extract of commercial L. nobilis had the highest AChE inhibition. The commercial sample of C. reticulata peel exhibited slightly better antioxidant and AChE inhibition activities. The origin of plant material was statistically significant in the majority of applied assays. The applied solvent was statistically significant in most cases, probably due to differences in the chemical composition of the extracts. The acetonic extract of L. nobilis had the highest TPC and also provided the best results in antioxidant tests. Since the acetonic extract of L. nobilis from Lastva showed the highest TPC, antioxidant activity and αglucosidase inhibition, and water extract of commercial L. nobilis exhibited the highest AChE inhibition, the bay leaves could be promising antioxidant, antineurodegenerative and antidiabetic agent.