Comparative Assessment of the Phytochemical and Selected Heavy Metal Levels in Cucumis sativus L. and Solanum aethiopicum L. Fruit Sample Grown in South Eastern and North Central Regions of Nigeria Respectively

Studies were carried out to comparatively assess the phytochemical and heavy metal levels in Solanum aethiopicum L. and Cucumis sativus L. fruit samples grown in the South Eastern and North Central regions of Nigeria respectively using standard analytical procedures and instrumentation. The fruit samples were assayed for selected heavy metals (Pb, Cd and Cu) using atomic absorption spectrophotometer after wet digestion of the samples. The five detected phytochemicals (flavonoids, alkaloids, terpenoids, saponins and glycosides) were present at varying amounts in the investigated fruit samples from the South Eastern and North Central regions of Nigeria. The range of mean values of flavonoids, terpenoids, saponins and glycosides in the S. aethiopicum L. fruit samples from the two studied regions were 0.50 - 0.57, 0.53 - 1.26, 0.44 - 0.78, 1.12 - 1.93 and 0.40 - 0.50 mg/g respectively. The range of mean values of flavonoids, only Cd was at toxic levels in the C. sativus L. fruit samples from the South Eastern and North Central regions of Nigeria. This is therefore a health concern to the fruit consumers that includes these fruits in their habitual daily fruit diets. Although the therapeutic and pharmaceutical benefits that would be derived from consuming, the investigated fruit samples has been shown in this study, there is therefore a possible risk of undue exposure to environmental pollutants such as heavy metal, especially through growing these fruits in polluted soils resulting from unwholesome anthropogenic practices. For fruit consumers to derive maximum health benefits from consuming these fruits, pollutants like heavy metals must be at non-toxic levels and this can only be achieved by ensuring that these fruits samples are grown and harvested in environments with less anthropogenic activities.


Introduction
Throughout the world from ancient times, fruits and vegetables have always formed an important part of diet for both human and animals [1]. Fruits and in general plants are sources of food to meet nutritional needs as well as some medicinal and therapeutic purposes.
Fruits and vegetables are consumed fresh or in processed form and known to be among the most important sources of phytochemicals for the human diet [2].
About 200,000 phytochemicals are known so far with about 20,000 of them have been identified as originating from fruits, vegetables and grains [3].
According to [4], phytochemicals are some of the most important natural preservation structures that help to reduce and inhibit pathogenic macro-organisms growth and preserve the overall quality of food products. [5] stated that dietary intake of phytochemicals may promote health benefits, protecting against chronic degenerative disorders, such as cancer, cardiovascular, neuro-degenerative diseases, diabetes, high blood pressure, inflamation, ulcers, osteoporosis, parasitic viral and microbial infections epidemiological and animal studies suggest that the regular consumption of fruits vegetables and whole grains reduces the risk of chronic diseases associated with oxidative damage [6].
Cucumis sativus L. is a widely cultivated plant of the gourd family which is eaten in the unripe, green form [7].
Its fruit extract has shown free radical scavenging and analgesic properties in 225 Natural Resources mice as reported by [8].
Studies by [9] have shown the anti-oxidant and anti-ulcer effects of C. sativus L. extracts in rats.
According to [7], C. sativus L. fruit contains several phytochemicals possessing antioxidant activity and the major groups of phytochemicals include vitamins A, C, E and K, carotenoids, tarpenoids, flavonoids, polyphenols, saponins, enzymes and minerals.
Solanum aethiopicum L. fruits can be eaten raw and also when boiled or fried, used as ingredient of stews, soups and vegetable sauces [10]. Wide variations exist within the vegetable and fruit characters both within and between the African egg species including variations of characters like diameter of corolla, petile length, leaf blade width, plant branching, fruit shape and colour [11]. The uses of S. aethiopicum L. fruits in indigenous medicine range from weight reduction to treatment of several ailments including asthma, allergic rhinitis, nasal catarrh, skin infections, rheumatic disease, swollen joint pains, gastro-esophageal reflux disease, constipation and dyspepsia [12].
The therapeutic and pharmacological importance of S. aethiopicum L. has been attributed to the presence of certain chemical substances such as phenols, anthocyanin, glycol alkaloids, flavonoids, saponins, ascorbic acid and a-chronine etc. [13]. Although heavy metals are naturally occurring elements, found throughout the earth's crust, most environmental contamination and human exposure arise from human activities such as mining and smelting operations, industrial production, domestic and agricultural activities [14].
Heavy metals are natural constituents of the environment, usually occurring in low concentration under normal conditions. Heavy metals contamination of vegetables and fruits cannot be underestimated as these food stuffs are important components of human diet [15].
Heavy metals can be readily taken up by vegetable and fruit roots and can be accumulated at high levels in the edible parts of plants. Anthropogenic activities can cause elevated levels of heavy metals in various parts of the ecosystem and may occur through various diffused and point sources [16].
According to [17] assessment of heavy metal compositions of vegetables and fruits is one of the most important methods used for monitoring environmental pollution as elevated levels of the elements in the human body is known to cause a number of serious metabolic and toxic discase disorders and effects.
Since fruits such as C. sativus L. and S. aethiopicum L. are known to be rich in secondary metabolites or phytochemicals that bring therapeutic effects to the body at regular consumption, geographical location of these fruits and the anthropogenic activities going on in such environment directly or indirectly plays a key role in determining the levels of phytochemicals as well as environmental pollutants such as heavy metals in these fruits.
Therefore, studies were carried out to assess the levels of phytochemicals and heavy metals in C. sativus L. and S. aethiopicum L. fruit samples grown in South

Sample Collection and Identification
The fruit samples of Cucumis sativus L. and Solanum aethiopicum L. were pur-

Preparation of Samples
Fruit samples of C. sativus L. and S. aethoiopicum L. from the differently studied environments were sliced into shreds and oven dried at 150˚C and then pulverized to obtain a fine dry powder.
The pulverized fruit samples were stored in air-tight containers at room temperature prior to analysis.

Quantitative and Qualitative Phytochemical Analysis of the Samples
Aqueous extract of the pulverized fruit samples of C. sativus L. and S. aethoiopicum L. were prepared by dissolving 20 g each of the samples with 70% ethanol in a 500 ml conical flask. The extracting mixture was allowed to stand for 24 hours and was subsequently filtered using Whatman filter paper No. 42 (125 mm).
The filtrate was afterwards concentrated in an oven at 80˚C [18]. The extracts of C. sativus L. and S. aethoiopicum L. fruit samples were tested for flavonoids, alkaloids, terpenoids, saponins and glycosides as described by [19].
Quantitative determination of glycosides and terpenoids were done using alkaline picrate method and oxidation method respectively as described by [19].
Saponin content was quantitatively estimated by spectrophotometric method of [20].
The flavonoid content was estimated using ferric chloride colorimetric methods of [21].
Quantitative determination of alkaloid content was carried out by oxidation method as described by [19]. Central regions of Nigeria respectively were determined using atomic absorption spectrophotometer (AAS) PG, 550 Model as described by [22].

Statistical Analysis
The data obtained was expressed as mean ± standard deviation and subjected to one way analysis of variance (ANOVA) at 5% level of confidence using SPSS version 22.0.

Flavonoids
Result of Table 1 Table 2.
[23] observed a moderate presence of flavonoids in the extracts of C. sativus L.
fruit samples from Uba Hong Local Government Area of Adamawa state which  According to [24], flavonoids plays important roles in biological activities including antiallergenic, antibacterial, antiviral and antifungal effects.

Alkaloids
Result of Table 1 shows that alkaloids had moderate presence in the S. aethiopicum L. fruit samples from South Eastern region while a trace presence of the phytochemical was observed in the ethanolic extracts of the fruit samples from the North Central region of Nigeria.
1.26 ± 0.10 and 0.53 ± 0.13 mg/g were the mean values of alkaloids in the S. aethiopicum L. fruit samples from the South Eastern and North Central regions of Nigeria respectively.
Additionally, alkaloids were observed to be present in trace amounts in the extracts of C. sativus L. fruit samples from the South Eastern and North Central regions of Nigeria respectively as represented in Table 2.
The mean values of alkaloids in the C. sativus L. fruit samples from the South Eastern and North Central regions of Nigeria were 0.55 ± 0.07 and 0.53 ± 0.04 mg/g respectively as shown in Table 2. [25] reported trace presence of alkaloids in S. aethiopicum L. fruit samples [10] stated that the bitterness of egg plants is due to the presence of alkaloids and that poisoning by Solanum species has been attributed to the presence of glycol-alkaloids which causes diarrhea.
According to [7], alkaloids and saponins are known to exhibit antispasmodic and antimicrobial activities, lower blood pressure, balance the nervous system and protects plants from microbial pathogens.

Terpenoids
Result of Table 1 shows that terpenoids were observed to have trace presence in  Table 2. Table 2 shows that the mean values of terpenoids in the C. sativus L. fruit samples from the South Eastern and North Central regions of Nigeria were 0.96 ± 0.14 and 1.51 ± 0.11 mg/g respectively. [23] reported a very high presence of terpenoids in the ethanolic extracts of C. sativus L. fruit samples collected from Uba Hong Local Government Area of Adamawa State, which completely agreed with the observed presence of the phytochemical in the C. sativus L. fruit samples from the studied North Central region of Nigeria.

Result of
[5] stated that terpenes have a unique antioxidant activity in their interaction with free radicals because it reacts with free radicals by partitioning themselves into fatty acid membranes by virtue of their long carbon side chain.
The most studied terpene antioxidants are the tocotrienols and tocopherols and are effective apoptotic inducers for human breast cancer cells [7].
The impact of a diet of fruit vegetables and grains on reduction of cancer risk may be explained by the actions of terpenes in vivo [26].

Saponins
Result of Table 1 shows that the ethanolic extracts of S. aethiopicum L. fruit  Table 1.
Also, saponins were observed to have moderate presence in the extracts of C. sativus L. fruit samples from the South Eastern region, where as the phytochemical had trace presence in the extracts of the fruit samples from the North Central region as represented in Table 2. 0.83 ± 0.04 and 0.41 ± 0.03 mg/g were obtained as mean values of saponins in the C. sativus L. fruit samples from the South Eastern and North Central regions of Nigeria respectively as given in Table 2.
[27] reported a moderate presence of saponins in C. sativus L. fruit samples from Nsukka, Nigeria, which compared very well with the observed presence of the studied C. sativus L. fruit samples from the South Eastern region of Nigeria.
However, [27] reported a higher mean value of 2.01 ± 0.08 mg/g for C. sativus fruit samples from Nsukka, Nigeria than it was obtained for the phytochemical in the fruit samples from the two investigated regions of Nigeria.
According to [27] saponins has the property of coagulating red blood cells, anti-inflammation, allelopathy, cholesterol-lowering.
[23] stated that saponins are known to be immune boosters.

Glycosides
Result of Table 1 shows that glycosides had trace presence in the extracts of S. aethiopicum L. fruit samples from the South Eastern and North Central regions of Nigeria respectively.
The mean values of glycosides in the of S. aethiopicum L. fruit samples from South Eastern and North Central regions of Nigeria were 0.40 ± 0.05 and 0.51 ± 0.06 mg/g respectively as shown in Table 1.
Also, glycosides were observed to have moderate presence in the C. sativus L.
fruit samples from the South Eastern region, where as the phytochemical had an observed very high presence in the fruit samples from the North Central region of Nigeria as represented in Table 2.
0.90 ± 0.8 and 1.74 ± 0.18 mg/g were the mean values gotten for glycosides in the C. sativus L. fruit samples from the South Eastern and North Central regions of Nigeria respectively as given in Table 2.
[27] reported a very higher mean value of 32.23 ± 0.41 mg/g for glycosides in C. sativus L. fruit samples from Nsukka, Nigeria, than what was obtained for the phytochemical in the C. sativus L. fruit samples from the two studied regions of Nigeria.
[23] reported a very high presence of glycosides in the C. sativus L. fruit samples from Uba Hong Local Government Area of Adamawa State, which com- pletely agreed with the observed presence of the phytochemical in the studied C. sativus L. fruit samples from the North Central region of Nigeria.
[6] stated that glycosides help to normalize blood pressure, balance blood sugar, lower cholesterol and blood lipids and improve liver function.

Lead
Lead is an extremely toxic heavy metal that disturbs various plants physiological processes and fastens the production of reactive oxygen species (ROS), causing lipid membrane damage that ultimately leads to the damage of chlorophyll and therefore suppresses the overall growth of the plant [17]. Table 3 shows that the mean levels of Pb in S. aethiopicum L. fruit samples from the South Eastern and North Central regions of Nigeria, were 0.24 ± 0.02 and 0.15 ± 0.08 µg/g respectively.

Result of
The mean levels of Pb in the S. aethiopicum L. fruit samples were within the [28] recommended permissible limits for the metal in edible food products.
The mean levels of Pb in the S. aethiopicum L. fruit samples from the South Eastern Region were observed to be statistically higher than that from the North Result of Table 4 shows that Pb had mean levels of 0.4 ± 0.05 and 0.31 ± 0.02 (µg/g) in the C. sativus L. fruit samples from the South Eastern and North Central  The mean levels of Pb in the fruit samples from the two studied regions were statistically significant and equally within the WHO recommended limits.
Although Pb was within permissible limits in the C. sativus L. fruit samples from the two studied regions, the mean values obtained showed intense contamination of the fruit samples with the metal and therefore a health concern especially on prolonged exposure to the metal through fruit diets.
[29] reported a mean value of 0.13 µg/g for Pb in S. aethiopicum L. fruit samples sold at Ologbo market in Benin City, Edo State, which compared very well with what this study obtained for Pb in S. aethiopicum L. fruit samples from North Central region of Nigeria.
[29] also stated that Pb is a harmful body poison that can enter into the human system through air, water and food and cannot be eliminated by fruits and vegetable washing.

Cadmium
Cadmium was only detected in the S. aethiopicum L. fruit samples from the South Eastern region of Nigeria with a mean value of 0.01 ± 0.00 mg/g as shown in Table 3. The mean value of Cd in the fruit samples was within the recommended permissible limits. 0.05 ± 0.01 and 0.02 ± 0.00 µg/g were the mean values obtained for Cd in C. sativus L. fruit samples from the South Eastern and North Central regions of Nigeria respectively as represented in Table 4.
The mean value of Cd in the C. sativus L. fruit samples from the South Eastern region was above the recommended permissible limits while the metal's mean value in the fruit samples from the North Central region was at the border line of the permissible limits. The levels of Cd in the C. sativus L. fruit samples from the two studied regions were statistically significant.
[29] reported a higher mean value of 0.845 ± 0.19 µg/g for Cd in S. aethiopicum L. fruit samples sold at Uselu market in Benin City, Edo state, than what this study obtained for the metal in the S. aethiopicum L. fruit samples from the South Eastern region of Nigeria.
According to [30], cadmium is a highly toxic non-essential heavy metal that is well recognized for its adverse influence on the enzymatic systems of cells, oxidative stress and for inducing nutritional deficiency in plants.

Copper
Result of Table 3 shows that the mean values of Cu in the S. aethiopicum L. fruit samples from the South Eastern and North Central regions of Nigeria were 0.78 ± 0.10 and 1.12 ± 0.16 µg/g respectively.
The levels of Cd in the fruit samples from the two studies regions of Nigeria were statistically significant and equally within the recommended permissible limits in edible food materials.
0.62 ± 0.08 and 0.96 ± 0.05 µg/g were the mean values obtained for Cu in C.  Table 4.
The levels of Cu in the fruit samples from the two studied regions differed significantly and were equally within the recommended permissible limits.
Copper is an essential micro nutrient required in the growth of plants and animals.
Excessive levels of Cu in the body lead to abnormal retention of Cu in the liver (Wilson disease) [14]. The mean value of 0.32 ± 0.08 µg/g reported by [29] for Cu in C. sativus L. fruit samples sold at Ologbo market in Benin City, Edo State, was lower than what this study obtained for the metal in the investigated C. sativus L. fruit samples from the two studied regions of Nigeria.

Conclusions
Flavonoids, alkaloids, terpenoids, saponins and glycosides were all found present although at varying amounts in the investigated fruit (Cucumis sativus L. and Solanum aethiopicum L.) samples from the two studied regions of Nigeria.
C. sativus L. and S. aethiopicum L. fruit samples from South Eastern region were observed to have moderate to very high presence of alkaloids, saponins, glycosides and flavonoids, where as the investigated fruit samples from the North Central region of Nigeria had moderate to very high levels of saponins, terpenoids, flavonoids and glycosides.
The varying amounts of the detected phytochemicals in the investigated fruit samples from the two studied regions of Nigeria, were attributed to differences in geographical location and soil chemistry, where the fruits were grown and harvested.
The presence of the detected phytochemicals in the investigated fruit samples from the two studied regions of Nigeria shows that consumption of these fruit samples by the people especially on regular basis would be of immense therapeutic and pharmacological importance.
The mean levels of Cd, Pb and Cu in the investigated fruit samples from the two studied regions of Nigeria were statistically significant.
Cd was at toxic levels in the C. sativus L. fruit samples from the two studied regions of Nigeria.
Taken into consideration the fact that Cd is a non-essential element and equally very toxic at very low concentrations, its levels in edible fruits, vegetables and food are of immense interest to scientists and environmentalists. It is important that fruits which usually form diets to people of all ages, are grown and harvested in environments with less anthropogenic activities and practices by the people that introduce excess pollutants into the soil should be checkmated by relevant authorities so as to prevent undue exposure to heavy metals through fruits consumption.