Nicotine content and Dietary composition of some cultivated Species of Solanaceae family

Despite the fact that vegetables are consumed in large quantities in our environment, there is a dearth of literature that focused on the optimum benefits to man especially in the management of disease like Parkinson's Disease (PD). Nicotine, the major phyto-constituent of Nicotiana tabacum of the family Solanaceae has been reported to be present in some other species of the family. This study investigated the nicotine content and the dietary components in the leaves and fruits of selected species of cultivated vegetable plants belonging to Solanaceae family. Edible plants from Solanaceae family were grown till the fruiting stage in the experimental farm of Ekiti State University, Ado Ekiti, while Nicotiana tabacum was used as the control plant. Plants samples from mature leaves and fruits of each plant were harvested washed with de-ionized water, oven dried at 30 o C. Powdered samples were analyzed for mineral and proximate components according to standard procedure and nicotine content were determined with GC-MS. Nicotine remained the major constituent of tobacco. Nicotyrine, a product of nicotine dehydrogenation was also detected in all the plant samples. Capsicum annum var. accuminatum fruit displayed the highest nicotine content (0.5543 mg/L) compared to the others with relatively lower quantities. Nicotyrine content was more available in the plant fruits than nicotine, with C. annum var abbreviatum displaying more of nicotyrine content (75.3456 mg/L). Solanum lycopersicum displayed the highest moisture contents (19.94 %), followed by tobacco leaf (11.88%). N. tabacum leaf displayed high quantity of protein (56.52%) than other members. The fruits of the solanaceae plant species were rich in carbohydrate, protein with very low amount of fat. This study revealed that the solanaceae plants contained more of nicotyrine than nicotine. In contrast to the previous studies, the nicotine content was higher in the eggplant leaves than the fruits. Eggplant and Capsicum species can be recommended for patient suffering from neurodegenerative diseases like PD


Introduction
Vegetables are considered essential for well-balanced diets in human food as they supply vitamins, minerals, dietary fiber, and phytochemicals associated with overall good health, improvement of gastrointestinal health and vision, reduced risk for some forms of cancer, heart disease, stroke, diabetes, anaemia, gastric ulcer, rheumatoid arthritis, and other chronic diseases (Hyson,2002).Each vegetable group contains a unique combination of phytochemicals in certain amount which distinguishes them from other vegetables within their own group (Dias, 2012).Among these phytonutrients is nicotine, which if consumed in moderate amount along with food, has been noted for having the capability of reducing the risk of developing Parkinson's Disease (PD) (Searles, 2013).Nicotine, the major phytochemical constituent of Nicotiana tabacum of the family Solanaceae has been reported to be present in some other species of the family (Castro and Monji, 1986;Sheen 1988).According to Leete (1983), about 12 families and 24 genera, including the nightshade family (Solanaceae) were identified as species containing nicotine to varying degrees.In their work, it was reported that nicotine is certainly present in human foods especially plants that belong to the Solanaceae family, many of which are significantly consumed by humans due to their nutritional values.
Nicotine is an alkaloid predominantly found in the leaves of Nicotiana tabacum, called tobacco plant, which belong to the Solanaceae family (Dias, 2012).Tobacco has been the major source of nicotine known worldwide because of its usage and consumption.However, perhaps due to its high contents of nicotine which has been known to have stimulatory effects on the central nervous system and cause addiction, there has not been much record of the nutritive value of the leafy plant.Hence, it has, therefore, not been used as edible dietary vegetable as some other members of the family.Although these vegetables are largely consumed in our local environments, not much research has been focused on the optimum benefits to man especially in the management of diseases such as PD.Apart from the fact that little information is known to people about the presence of nicotine in variable amounts in various few reported vegetables of the Solanaceae family consumed in different parts of Nigeria, only a few citations can be found in the literature that address nicotine concentration in diverse foods and the ensuing dietary intake of nicotine.Therefore, this study finds it important to investigate the nicotine content and the dietary components in the leaves and fruits of selected species of cultivated vegetable plants belonging to Solanaceae family, popularly consumed among the Nigerian population.

Chemicals
Nicotine standard was purchased from Sigma-Aldrich Company with purity 99.9%.Methanol (BDH, UK), dichloromethane and de-ionized water were of HPLC grade.Anhydrous sodium sulfate (Merck, Germany) was activated by heating at 200 °C before use.Silica gel (60-120 mesh, Loba, India) was activated at 400 °C for 12 h prior to use.

Experimental Design
The experimental set up was arranged in Randomized Complete Block Design with 10 replicates for each of the plant species.

Plant Materials
Edible plant samples from Solanaceae family which include Capsicum frutescence var baccatum (Ata wewe), Capsicum annum var abbreviatum (Ata rodo), Capsicum annum var acuminatum (Ata bawa), Solanum macrocarpon, Solanum aethiopicum and Solanum lycopersicum were grown till the fruiting stage in the experimental farm of Ekiti State University, Ado Ekiti, Ekiti State while Nicotiana tabacum used as the control plant, was harvested from a nearby farm in Ado Ekiti.After collection, the samples were kept in a polyethylene bag with aluminum foil protected cover and stored in refrigerator at 2 °C to avoid any deterioration.Mature leaves and fruits from each plant samples were harvested, washed with de-ionized water to remove dusts and any other foreign particles.Samples were thereafter oven dried at a low temperature of 300C and blended into fine powder.
Precaution against Possible Sources of Contamination.Special emphasis was put on possible contamination sources of nicotine from the air or any equipment used.Smoking was not permitted around the experimental farm and in the laboratory area.Preparation of samples for GC-MS Quantitative Determination of Nicotine.
Finely ground samples of the fruits and leaves of each species were used for the GC-MS quantitative determination of nicotine contents.This was prepared by dissolving 0.1g of each sample into 1mL of methanol to give 0.1 g/ mL (0.1 g/mL ≡ 100 mg/mL ≡ 100,000 mg/L or 100,000 ppm).The experiments were carried out in the laboratory of the Department of Biological Sciences, Afe Babalola University, Ado-Ekiti.

Proximate Composition
Proximate compositions of powdered samples were determined according to the procedure of Association of Official Analytical Chemists (AOAC, 2010).For moisture content 2.0g of the sample(s) were placed in an oven maintained at 100 -103°C for 16 hours with the weight of the wet sample and the weight after drying noted.The drying was repeated until a constant weight was obtained.The moisture content was expressed in terms of loss in weight of the wet sample.Ash content was determined by taking 2.0g of each of the oven-dried samples in powder form were accurately weighed and placed in crucible of known weight.These were ignited in a muffle furnace and ashed for 8 hours at 550°C.The crucible containing the ash was then removed, cooled in a desiccator and weighed and the ash content expressed in term of the oven-dried weight of the sample.Protein, fiber, and fat were analyzed following the same standard procedure while carbohydrate content was determined by ding together the sum of the percentage moisture, ash, crude lipid, crude protein and crude fiber, then subtracting this value from 100.Analysis was carried out in triplicates and all values were reported in percentage.The instrument used was auto-zeroed using the blank (distilled water) for each element, and the standard was aspirated into the flame from the lowest concentration to the highest concentration.Corresponding absorbance was taken and the graph of absorbance against concentration was plotted.The samples were analyzed with the concentration of metals being shown in parts per million (ppm) after extrapolation from the standard curve (Greenberg et al., 1985).

Estimation of Mineral Composition
The analysis of metal concentration in the samples was determined by weighing one (1)g of each of the ground samples into different platinum crucibles that had been well labeled.It was then placed in a muffle furnace at 450 0 C -550 0 C until all of the carbon present was removed as evidence by white ash.Thereafter, 2mL of distilled water + 1mL of concentrated HNO3 + 1mL of concentrated HCl was added and was warmed gently so as to speed up the dissolution of the ash.The dissolved ash was then brought up to 10mL.The metal concentration was determined using Atomic Absorption Spectrophotometer (AAS) (Buck Scientific model 210 VGP) by calibration method.Three processes were involved which are: standard preparation, equipment calibration and sample analysis.

Statistical analyses
Data were expressed as means of triplicate measurements.Correlations were obtained by Pearson correlation coefficient in bivariate correlations.Means were compared by Tukey-HSD and LSD (least significant differences).Differences at P < 0.05, were considered to be significant.

Babawale et al., 2022
Nicotine content and Dietary composition of some cultivated Species 42| This journal is © The Nigerian Young Academy 2022 Annals of Science and Technology 2022 Vol.7 (2) 42-50

Results
Table 1: GC-MS Quantitative Analysis of Nicotine and Nicotyrine Concentration in the Fruits of Cultivated Species of Selected Solanaceae Family and the leaf of Nicotiana tabacum Values depict variations in the nicotine and Nicotyrine contents.The result showed that N. tabacum, the standard plant revealed more of nicotine (4477.68mg/L) than nicotyrine (671.74 mg/L).Among the fruit samples, C. annum var.accuminatum fruit displayed the highest nicotine content (0.5543 mg/L) compared to the other member of the family with relatively lower quantity.C. annum var abbreviatum displayed more of nicotyrine content (75.3456 mg/L) as compared to other fruits which altogether contained an appreciable lesser amount as compared to control plant Table 2 shows that nicotine and nicotyrine were discovered to be present in all the plant samples.Values depict variations in the nicotine and nicotyrine contents of the leaf samples.Solanum aethiopicum leaf also known as eggplant showed abundance of nicotine (32.19 mg/L), present in its leaves as compared to leaves of other species with relatively lower amount of nicotine when compared with the nicotine and nicotyrine content of Nicotiana tabacum (4477.68mg/L and 671.74 mg/L, respectively).

Discussion
Accumulating evidence had suggested that nicotine may be of therapeutic value of Parkinson's disease.In this investigation, Nicotiana tabacum leaf was used as standard plant which is already known for its high content of nicotine (Serban et al., 2016).The result of the GC MS analysis confirmed that tobacco plant is highly rich in nicotine content (Table 1 and Table 2).Apart from nicotine, the major constituent of tobacco, nicotyrine, a product of nicotine dehydrogenation was also detected in all the plants including the standard plant itself.Nicotyrine is described as a product of nicotine dehydrogenation in e-vapor and is a known inhibitor of human cytochrome P450 enzyme, which mediates nicotine metabolism (Yeongkwon et al., 2018).Nicotine oxidation during storage was reported capable of forming nicotyrine (Wada et al., 1959).The discovery of nicotyrine in the selected plants of this present study makes it a novel observation which may require more attention for further research.While nicotyrine was found to be more detected in the sample plants than nicotine, nicotine was detected in trace amount in all the sampled plants, from Solanum genus including tomato, eggplant, and from Capsicum genus, when compared with the control plants.The reverse was the case in N. tabacum which revealed more of nicotine than nicotyrine.Nicotine has been known for its various pharmacological functions.It is a known drug that stimulates nicotinic acetylcholine receptors implicated of having therapeutic value on PD (Maryka et al., 2008).The detection of nicotyrine in these plants might serve an added advantage in the bioactivity of this plant family and in the management of some medical challenges especially Parkinson's disease which has been linked to consumption of edible vegetables that contain nicotine in trace amount.
According to the literature, apart from Nicotiana species, other members of the Solanaceae family whose edible fruits and tubers including peppers, tomatoes, eggplants and potatoes have been implicated of containing nicotine in lower concentrations (Castro and Monji, 1986;Sheen, 1988;Domino et al., 1993;Siegmund et al., 1999;Searles et al., 2013).Most of the few studies were carried out on the fruit of these plants except the work of Serban et al., (2016) who reported on the tomato leaf which is not often consumed.The fact that the fruits parts of the members of solanaceae family are commonly consumed as food, special interest was placed on investigating the nicotine and dietary contents of the leaf portion of these plants which are not mostly consumed as food.The presence of nicotine in trace amount in the selected plants of this study confirmed the reports that the amount of nicotine absorbed from these foods is considered negligible relative to the amount obtained from active smoking, and probably lower than that from environmental tobacco smoke (Andersson et al., 2003), hence, their therapeutic properties without causing any stimulation to the body.According to the work of Searles et al., (2013), carried out on fresh and processed vegetables, it was stated that vegetable consumption in general did not affect Parkinson's disease risk, but as consumption of edible Solanaceae species increased, Parkinson's disease risk decreased with peppers displaying the strongest association.Andersson et al., (2003) also reported that the level of nicotine in these vegetable plants was by observation varied both among the various plants and between processed and fresh products.
The result of this study agreed with the growing reports which stated that the consumption of certain food groups like the member of Solanaceae family of plants are associated with health benefits and may contain promising components in them that may elicit neuroprotection in PD (Evatt, 2007, Searles et al., 2013;Shaltiel-Karyo et al., 2013).Also, phytochemicals, vitamins, minerals present in the food supplements or fruits, vegetables, and spices are capable of preventing, delaying, or alleviate the clinical symptoms of chronic neurodegenerative diseases, improving cognitive functions, learning, general brain status, and wellbeing (Olasehinde, et al., 2017).Seid et al., (2014), reported that dietary intake of nicotine-containing vegetables from edible Solanaceae including tomatoes, potatoes, and peppers, was associated with a reduced risk of PD in men and woman who had never smoked cigarettes or tobacco (Searles et al., 2013).However, it remains unclear as to whether the observed protective effect was due to the nicotine content or other components of this group of vegetables especially the dietary components.Further investigation about the dietary components revealed that the proximate composition of the plant samples as shown in Table 3 and  Table 4 were very rich in carbohydrate, protein and crude fiber with a very low amount of fat.Regulation of intake of carbohydrates, protein, fluids and fibers and some other nutrients present in fruits and vegetables have been considered to be very important in the nutritional and functional decline associated with aging and may slow the progression of P.D (Liu, 2003;Mostafavi and Hosseini, 2015;and Michele et al., 2019,).The presence of fat content at low quantity in all the plants suggests that the fruits and the leaves of these species had more of carbohydrates and proteins than that of fats.The role that fat plays in PD was considered most likely to be related to the type of fat in the patient's diet (De Lau et al., 2005), the patient's HDL/LDL ratio, total cholesterol levels and genetic factors.There is need to clarify the associations between fat intake and PD by conducting a large randomized controlled study.Inadequate fat in the body could result to body rashes, skin dryness, air loss and weaker immunity therefore, may require the compliment of other supplements to balance the diet.Interestingly, the control plant's leaf, Nicotiana tabacum, had higher protein content than the fruits and leaves of other members of the family.The high protein content of this plant might place a new significant importance on tobacco leaf.Previous studies have highlighted some of the health benefits associated with the consumption of Capsicum spp.The leaves of Capsicum frutescens var baccatum had the least protein content.The highest value of carbohydrate was contained in the fruits of Capsicum annum var abbreviatum when compared to other samples while the leaves of tobacco had the lowest content of carbohydrate.More so, fibre was abundant in the fruits of Capsicum frutescens var baccatum followed by Solanum aethiopicum leaf when compared to the standard plant Nicotiana tabacum leaves which had the least crude fiber content.Ogunruku et al., (2014) reported the two pepper varieties Capsicum annuum var.accuminatum and Capsicum chinense, implicating them of their inhibitory effect on acetylcholinesterase and butyrylcholinesterase activities and also antioxidant properties of Capsicum spp.Dietary fiber plays an important role in the maintenance of good intestinal and good digestive health, prevention of cardiovascular disease, some types of cancer, peptic ulcer, improve skin health and reduce the risk of developing obesity, and diabetes mellitus (Buttriss, 2008).It is indicative that these vegetables including other member of the family are capable of possible dietary means by which oxidative stress and symptomatic cognitive decline associated with neurodegenerative conditions could be alleviated.
Table 5 and Table 6 showed the result of the mineral composition of the leaves and the fruits of the selected plant samples.Nearly all the Solanum spp and Capsicum spp are very rich in Na, K, Ca and Mg.The result revealed that the plants are very good source of mineral elements.This result has by indication showed that the Solanum species contain less of all the mineral contents when compared to the Capsicum species except for the Solanum lycopersicum fruit which is very rich in potassium just like the Nicotiana tabacum leaves.Tomato (Solanum lycopersicum fruit) and peppers (Capsicum spp.) have a worldwide distribution.They are important as vegetable foods and spice and are considered an important source of nutrients in the human diet which are grown in tropical, subtropical, and temperate regions.Various species and varieties occurring within the Capsicum genus depending on their shape, size, flavor, and hotness include Capsicum annum, Capsicum frutescence, and Capsicum chinens (Loizzo et al., 2008, Tundis, 2011).Unlike vitamins, there is little or no literature on the importance of mineral elements in the management of PD.This result becomes so important when the Babawale et al., 2022 Nicotine content and Dietary composition of some cultivated Species 48| This journal is © The Nigerian Young Academy 2022 Annals of Science and Technology 2022 Vol.7 (2) 41-50 usefulness of such essential minerals like Ca, Na, K, Mg, and Fe are all present in the fruits and the leaves of the sample species and they play important role in the management of PD.All the Capsicum species are very good source of all the minerals in a very good and appreciable amount.Copper and zinc are the essential trace elements that are needed only in minute quantity by the human body for important biochemical functions.It is not surprising that all the samples contain these two elements in trace amount which is an indication why they are considered important spices in various diets.This suggests their importance as the major source of vegetable to mankind.No traces of lead was determined in the S. aethiopicum F, Capsicum annum var abbreviatum F, C. frutescens var baccatum F, and Nicotiana tabacum L.

Conclusion
This study revealed that the Solanaceae plants contain more of nicotyrine than nicotine which called for attention to focus on its pharmacological properties in further studies.The nicotine content is more present in the eggplant leaves than the fruits which are contrary to some previous studies.Nicotiana tabacum leaves was also discovered to be very rich in protein therefore could serve a health benefit if the nutritional properties are more explored.It can be concluded that consumption of eggplant and Capsicum species can be of great benefit to the human health and can be specially recommended for patient suffering from neurodegenerative diseases like PD.

Conflict of Interest
Authors have declared that no conflict of interests associated with this study or any of the procedures and materials used for the purpose of the study.

Figure 1 :Fig 3 :
Figure 1: The chromatograph showed the retention time of nicotine and nicotyrine.The nicotine had a retention time of 8.843 mins and Nicotyrine had the retention time of 11. 85 mins.

Table 2 :
GC-MS Quantitative Analysis of Nicotine and Nicotyrine Concentrations in the Leaves of Cultivated Species of Selected Solanaceae Family and the leaf of Nicotiana tabacum

Components Concentration in the samples (mg/L) Solanum lycopersicum S. aethiopicum S. macrocarpon C. annum var abbreviatum C. annum var accuminatum C. frutescens var baccatum Nicotiana tabacum
Babawale et al., 2022 Nicotine content and Dietary composition of some cultivated Species This journal is © The Nigerian Young Academy 2022 Annals of Science and Technology 2022 Vol.7 (2) 41-50 | 43

Table 3 :
Proximate composition of the Fruits of Cultivated Solanum Species.
Values are mean ± standard deviation of triplicate determination.The result in Table3showed that all the fruits of the solanaceae plant species are rich in carbohydrate, protein with very low amount of fat.N. tabacum leaf displayed highest quantity of protein (56.523) than other members of the family.Solanum lycopersicum displayed highest moisture contents (19.94 %), followed by tobacco leaf (11.88%).

Table 4 : Proximate composition of the Leaves of Cultivated Solanum Species.
Values are mean ± standard deviation of triplicate determination.The result in Table4showed that all the leaves of the solanaceae plant species are rich in carbohydrate, protein with very low amount of fat.N. tabacum leaf displayed high quantity of protein (56.52%) than other members of the family.The leaves of Capsicum frutescens var baccatum had the least protein content (33.91%).

Table 5 : Mineral Composition in the Fruits of Cultivated Solanum Species
Values are mean ± standard deviation of triplicate determination.ND = Not detected Table5shows the mineral composition present in the fruits of cultivated species of selected Solanaceae family.Values depict the mineral contents present in the fruits of the samples.No traces of lead was determined in the S. aethiopicum F, Capsicum annum var abbreviatum F, C. frutescens var baccatum F, and Nicotiana tabacum L.

Table 6 : Mineral Composition in the Leaves of Cultivated Solanum Species .
Values are mean ± standard deviation of triplicate determination.ND = Not detected Table6shows the mineral composition present in the leaves of cultivated species of selected Solanaceae family.Values depict the mineral contents present in the fruits of the samples 46| This journal is © The Nigerian Young Academy 2022 Annals of Science and Technology 2022 Vol.7 (2) 41-50