Effect of seasonal changes on the quantity of secondary metabolites from neem and eucalyptus plants in North Central Nigeria

Traditional healthcare system depends majorly on natural medicinal plants from the environment. These plants produce secondary metabolites which confer on them the various medicinal properties; however, Seasonal fluctuations have impact on their availability and quantity hence their therapeutic efficacy. This study was carried out to evaluate the effect of seasonal changes on the quantity of secondary metabolites from Neem and Eucalyptus plants. Leaves and bark of Neem and Eucalyptus plants from SHESTCO, Federal Capital Territory (FCT), Abuja, Nigeria, were collected during the four quarters of the year 2019. Samples were dried, milled into powder, quantity of secondary metabolites was estimated and antioxidant activity was analysed using standard methods and protocols. Results from this study showed a variation in secondary metabolite compositions in response to seasons. In both plants’ organs, saponin content peaked and crashed during the second and fourth quarters of the year respectively, while alkaloid had the highest content during the fourth quarter of the year. Highest level of tannin was recorded in the leaf and bark of the plants during the third quarter of the year. Antioxidant activity of both plant extracts showed a regular patterned decrease with increasing concentration, with lowest antioxidant activity for both plants’ organs recorded during the third quarter. Findings of the study indicate that the quantity of inherent secondary metabolites in the medicinal plants and their corresponding antioxidant activity varies in specific manners at different times of the year due to seasonal variation.


Introduction
Medicinal plants are highly valuable to human livelihood. WHO estimated that 60% of the world population and 80% of the population of developing countries rely on traditional medicine, mostly plant drugs, for their primary health care needs [1]. Medicinal plants synthesize a number of biological constituents referred to as secondary metabolites or phytochemicals. These are needed for the plant's survival and its protection against predators and insects. The secondary metabolites are an extremely diverse group of compounds not only chemically but also in their functions where they are commonly used in agriculture and drug discovery [2][3][4]. The medicinal value of these plants lies in their bioactive phytochemical or secondary metabolite constituents that produce definite physiological action on the human body [5].
There is a marked variation in secondary metabolite constituents of medicinal plants due to a number of environmental variables such as temperature, altitude, soil type and change of season/rainfall [6][7][8][9][10][11]. The production of a variety of phytochemicals is the most successful adaptation of plants while developing various physiological mechanisms by which they were able to face both biotic and abiotic stress and threats [12][13][14]. The production of secondary metabolites, thus, affords plants, unlike other organisms, the ability to survive different seasonal conditions without hibernation [2]. Seasonal changes expose plants to different temperature levels (including extreme levels) that have an effect on their phytochemical compositions, with volatile compounds being the most affected [15]. There are several assumptions regarding the time and season for the collection of various parts of the medicinal plants like spring is suitable for the collection of bark, winter for essential oils etc. [16]. It has been reported by many researchers that during seasonal changes, the content of phenolics and flavonoids varied with the developmental phase of the plants. Djurdjevic and coworkers [17] observed that the maximum content of total phenolics was found in Conyza Canadensis L. plants during the flowering and fruiting time in August rather than phenolics which peaks during elongation and excessive plant growth in May and June or in September. In a study on the effects of seasonal variation of the major secondary metabolites present in the extract of Eremanthus mattogrossensis (Asteraceae) leaves, a straight correlation between the quantity of metabolites and seasonality was indicated, suggesting that environmental properties elicit important metabolic responses [18].
Eucalyptus globulus and Azadirachta indica have been reported to be rich sources of phytochemical compounds such as flavonoids, alkaloids, tannins and propanoids, extracted in the leaf, stem and root of the plants [19][20][21][22]. Their antioxidant property has also been widely reported [23][24][25]. Determination of the seasonal effect on plant phytochemical compositions provides knowledge on the time/season of harvest of individual plant species that afford optimum concentration of active ingredients [26]. Thus, this study aimed at investigating the variation in the yield of Azadirachta indica and Eucalyptus globulus bark and leaves across the seasons of the year as well as evaluating if the variation of these metabolites modifies the antioxidant activity of the extract from the studied plants' parts.

Material and methods
The bark and leaves of Azadirachta indica and Eucalyptus globulus were collected from the premises of Sheda Science and Technology Complex (SHESTCO) in Sheda village, Kwali, Abuja, located in the North Central region of Nigeria; 8.87 o North latitude, 7.01 o East longitude and 197 meters elevation above sea level. The collection of samples was done during the dry and rainy seasons. The dry and hot period in march 2019 with average temperature of 29.6°C, average rainfall 20 mm, average humidity 38% and the dry and cold period (harmattan) in December 2019 with average temperature 26.7°C, average rainfall 4 mm, average humidity 26%. The normal rainy period of June 2019 with average temperature 24.5°C, average rainfall 205 mm, average humidity 82% and the heavy rainy period in September 2019 with average temperature 23.6°C, average rainfall 290 mm, average humidity 85%. Temperatures and rainfall levels data were obtained from climatedata online [28] for the period January to December, 2019. The leaf samples were dried at room temperature, ground to powder and stored in the dark until further usage.

Phytochemical analyses
The bark and leaves of both medicinal plants neem and eucalyptus were analyzed for the quantity of simple phenols, flavonoids, tannins, alkaloids, and saponins, using standard procedures for the quantitative analysis of these phytochemicals.

Determination of Saponin
Estimation of the quantity of saponin was carried out as described by Obadoni and Ochuko [28]. 0.5g of the sample was added to 20ml of 1N HCl and was boiled for 4h. After cooling, it was filtered and 50 ml of petroleum ether was added to the filtrate and it was evaporated to dryness. 5 ml of acetone-ethanol was added to the residue. 0.4 ml of each was taken into 3 different test tubes, 6 ml of ferric sulphate reagent was added into them followed by 2 ml of conc. H2SO4 acid. The mixture was thoroughly mixed after 10 mins and the absorbance was taken at 490nm

Determination of flavonoids
The determination of flavonoids in the samples was done by acid hydrolysis method as described by Vabkova and Neugebauerova [29]. 0.5g of dried samples was mixed with 5ml of dilute hydrochloric acid and boiled for 10 mins. The boiled extract was allowed to cool and filtered. 1 ml of the filtrate was added to 5 ml of ethyl acetate and 5 ml of 1% ammonium. This was then scanned from 420-520nm for the absorbance.

Determination of Phenol
The quantity of phenol was determined using the spectrophotometric method as described by AOAC (1995). 0.5g of sample was boiled with 50ml diethylether for 15mins. 5ml of the boiled sample was then pipette into a 50ml flask and 10ml of distilled water is added. 2ml of ammonium hydroxide solution and 5 ml of boiled conc. Butanol was then added to the mixture. The mixture was made up to the mark and left for 30mins to react for colour development and absorbance measured at 505nm wavelength using a spectrophotometer.

Determination of alkaloid
Determination of alkaloid was done by procedure described by Manjunath and colleagues [31]. 0.5g of leaves sample was macerated with 20ml 96% ethanol and 20% Tetraoxosulphate (VI) acid (1:1v/v). The filtrate (1ml) was added to 5ml of 60% Tetraoxosulphate (VI) acid and allowed to stand for 5 mins. 5 ml of formaldehyde in 60% H2SO4 was then added and allowed to stand for 3 hrs. The absorbance was read at 565nm.

Determination of Tannin
The quantity of tannins in the samples was determined by using the spectrophotometric method as described by Van-Burden and Robinson [32]. 0.5g of the samples was weighed into a conical flask. 50 ml of distilled water was added and stirred for 1 hr. The sample was filtered into a 50 ml volumetric flask and made up to mark. 5 ml of the filtered sample was then pipetted out into a test tube and mixed with 2 ml of 0.1M HCl and 0.008M K4Fe(CN)6.3H2O. The absorbance was then measured with a spectrophotometer at 395 nm wavelength within 10 mins.

Determination of Antioxidant Activity
The antioxidant activity of the samples was evaluated by discolouration of 2, 2-diphenyl-1-picrylhydrozyl radical (DPPH) in methanol by a slightly modified method of Brand-Williams et al. [33]. The following concentrations of the extract were tested (0.1, 0.3, 0.5, 0.7, 1.0 mg/ml). The absorbance was monitored at 517nm. Vitamin C was used as the antioxidant standard at concentrations (0.1, 0.3, 0.5, 0.7, 1.0 mg/ml). A blank solution was prepared containing the same amount of methanol and DPPH. The radical scavenging activity was calculated using the following formula: Where Ab = Absorbance of the blank As = The absorption of the sample extract.

Statistical analysis
Experimental results were expressed as means ± standard deviations (n = 3, from 3 independent experiments). Differences between phytochemical quantities of the plant leaf and bark samples were determined using analysis of variance (ANOVA, SPSS). The values were regarded as statistically significant at p < 0.05.

Quantitative Phytochemical Analysis
Determination of the effect of seasonal changes on the quantity of plants phytochemicals is an important step to establish the suitable harvesting season with high amounts of phytochemicals. Seasonal quantitative phytochemical analysis of the bark and leaves of Azadirachta indica and Eucalyptus globulus showed that these medicinal plants have different alkaloids, flavonoid, phenols, tannin, saponins and tannins content during various seasons of the year. The results are shown in Figures 1-5 for alkaloid, flavonoid, phenol, saponin and tannin respectively for Azadirachta indica and Eucalyptus globulus bark and leaves.

Alkaloids Content
The quantity of alkaloid obtained from the leaf and bark of Eucalyptus and Neem plants was significantly high during the cold period, fourth quarter. There was no significant difference in the alkaloid content from the plants' samples during first and third quarter except from leaf of Eucalyptus globulus. The result showed that alkaloid quantity from the bark was significantly higher than that obtained from the leaves of both plans (Figure 1).

Phenol content
The phenol quantity obtained from Azadirachta indica and E. globulus leaves was significantly high during the hot period, first quarter, of the year. A significantly high quantity was obtained during the first and second quarter of year from neem bark while a significant quantity was obtained from the bark of E. globulus during the first and fourth quarter.
The lowest quantity of phenol was obtained during the third quarter from the plant samples ( Figure 3). The leaves of both planted revealed high quantity of phenol when compared to the bark.

Discussion
The results from this study indicate that medicinal plants may have high accumulation of different phytochemicals during different seasons, as seen in the case of alkaloids and tannins been accumulated in high amounts during colder seasons while flavonoids were accumulated in high amounts during warmer seasons for the medicinal plants Azadirachta indica and Eucalyptus globulus. The results obtained from this study are in agreement with the findings of the previous studies in other plants. Gololo and colleagues reported that high amount of alkaloid is usually obtained from medicinal plants during the colder season [3]; the leaves of Barleria prionitis, Boerhavia diffusa, Citrillus colocynthis and Grewia tenax were reported to have different yields of total alkaloids and polyphenols in different seasons, with the Indian summer (April-June) showing the highest yield compared to winter (December-February) and rainy season (July-September) [34]. In a separate study, total alkaloids and polyphenols were high during summer season in Convoliulus microphyllus, whereas the same phytochemical groups were high during winter in Datura metel [35]. Galolo et al. [3] reported that the leaves of Barleria dinteri, Grewia flava and Jatropha lagarinthoides, collected from Limpopo province in South Africa displayed high yield of simple phenol during Autum (March) in G. Flava and during summer in Jatropha lagarinthoides. The differences in the seasonal phytochemical quantities of the leaves of medicinal plants were earlier reported to cause seasonal variation in their biological activities [36].
The four seasons during which plant samples were collected are characterized by different factors such as water level in the soil, evapotranspiration rate, light intensity, photosynthetic efficiency, plant water potential and plant stage, directly respond to these variations [3,[37][38][39]. Ncube et al. [40] have found variation in the production of polyphenols in Tulbaghia violacea, Hypoxis hemerocallidea, Merwilla plumbea and Drimia robusta in different seasons, the explanation lies precisely in the climate differences, biotic and environmental conditions in addition to genetic factors. Other authors [41][42][43][44][45][46][47] have also attributed the variation in the production of secondary metabolites to environmental factors.
Both plants' bark and leaves were shown to have lowest antioxidant activity during the cold period, third quarter, of the year while the highest antioxidant activity was observed during the first quarter for both plants' leaves and bark. This report is in agreement with previous reports by Ramírez-Briones et al. [48] and Aoussar et al. [49] who reported that high antioxidant activity was recorded for plants such as Pseudevernia furfuracea, Evernia prunastri and Ramalina farinacea from Morocco during winter (March) and spring (May). Previous studies by Said et al. [24] and Tálos-Nebehaj et al. [50] have reported a correlation between the quantity of phenol and antioxidant capacity of plants. They established that polyphenols are primarily responsible for the antioxidant properties of plants. Thus in this study, a low antioxidant capacity and significantly low quantity of phenol was recorded during the cold season for the plants' leaves and bark while a significant high quantity of phenol obtained during the hot period of the year correspond with the high antioxidant capacity recorded during this period as well.

Conclusion
Taken together, the findings of the study indicate that the quantity of inherent secondary metabolites of the leaves and bark of Eucalyptus globulus and Neem plant and their corresponding antioxidant activity varies in specific manners at different times of the year due to the seasonal variation. Determination of the effect of seasonal changes on the quantity of plants phytochemicals is an important step to establish the suitable harvesting season of the plants in order to obtain high amounts of specific phytochemicals for medicinal purposes. Thus, from our study we recommend the cold period as the optimum period for the use of alkaloids and tannin from Azadirachta indica and Eucalyptus globulus for medicinal purpose, due to the high yield during this period.

Acknowledgments
The Authors are thankful to the management of Sheda Science and Technology Complex (SHESTCO) for making the laboratories available for use, and the supply of some reagents used for the study.