Chemical divergence of the Juglans Regia L. across districts Swat and Dir, Khyber Pakhtunkhwa, Pakistan

Juglans regia L. are nutritious fruit bearing plants mostly found in Northern areas of Pakistan. The population of walnuts was explored from district Dir and Swat Khyber Pakhtunkhwa, Pakistan for their geographical, climatic and chemical divergence. The geographical differences such as altitude, latitude and longitude whereas climatic differences viz. soil EC, soil pH, precipitations, intensity of light, temperature and soil temperature. In both districts TPC ranged from 211.2±0.6 to 227.8±0.4 mg/100g, RSA ranged from 43.32±1.5% to 52.18±0.4%, conductivity ranged from 296.43±0.6 to 312.22±0.3 S/m and elemental composition such as iron, copper, calcium, zinc and magnesium in Dir differs from 0.312±0.032, 0.209±0.13, 20.0±0.313, 0.406±0.10 and 10.2±0.030 mg/L to 0.543±0.65, 0.698±0.82, 28.7±0.234, 0.685±0.15 and 17.6±0.015 mg/L respectively. Altitude and temperature showed a correlation with total phenolics contents and radical scavenging activity while soil pH, precipitations, soil temperature, soil Ec and light intensity indicated a weak correlation with chemical traits of walnuts. Further studies of walnuts are needed to explore their therapeutically important phytochemicals to succeed naturally pharmaceutical nutrients of the maximum significance for the health of human beings. Ec


Introduction
From earlier ages walnuts were used worldwide for humans as a source of nutrition. Their kernels contain more proteins and oil contents that make their fruit best for human health. In the FAO list walnuts for human nutrition is classified as a strategic species and it counted as a central concern plant (Gandev, 2007). Walnuts are rich in nutrients food stuff because it has vitamins, minerals fats, and proteins. It is also a good source of phenolic acids, flavonoids, sterols, pectic substances and interrelated polyphenols. Walnuts nutritive contents vary from cultivar normal protein assessment that is up to 18.2% including 70% of the entire seed protein along with fewer amounts of the albumins (7%) globulins (18%) and prolamin about 5%. The proteins of walnuts contain all essential amino acids that are required for our healthy life. New taxa were added to the flora of Pakistan that had highly medicinal importance (Ali et al., 2017). Most of the medicinal plants showed highly antibacterial and antifungal potentials (Ullah et al., 2018;Iftikhar et al., 2019;Irfan et al., 2021Irfan et al., , 2024Ullah et al., 2022).
Amino acids exist in precise proportion in the walnuts especially the lysine and arginine ratio. Low lysine and arginine proportion declines atherosclerosis enlargement (Martinez et al., 2010). Similarly, walnuts contain a proper number of vitamins and minerals like magnesium (Mg), iron (Fe), Phosphorus (P), and Potassium (K) (Cosmulescu et al., 2009). Copper (Cu), Calcium (Ca), Sodium (Na), and Zinc (Zn) are present in accurate ratio (Siahnouri et al., 2013). Macronutrients are significant for the accurate development and growth of organisms (Jeszka-Skowron et al., 2017;Ilyas et al., 2020Ilyas et al., , 2022. Various macronutrients are involved in enzymatic response for the overall absorption of the organisms. The micronutrients help in the maintenance of heart diseases, bone function, immunity system, and nervous body function (Abdel-Aziz et al., 2016;Irfan et al., 2019b). The aim of the present study was to investigate the total phenolic contents, radical scavenging activity and some minerals contents of the newly selected walnuts genotypes from the Northern areas of Pakistan.

Selection procedure
Forty-four mature walnuts plants were labelled in different localities of district Swat and Dir in January 2018. Walnuts plants were occurring naturally in the hilly region of district Swat and Dir. The location's genotype's name was based on their spot and designated by the alphabetical abbreviation. Selected plants were mature in cropping condition and healthy.

Nuts collection
From walnuts, nuts were collected from different locations of the district Swat and Dir in October 2018.
to cultivar which may be affected by their genotype, different ecosystem, soil conditions, and cultivator (Muradoglu et al., 2010). The oil of walnut has several constituents that are α-linolenic acids, triacylglycerols (980 g/kg), linoleic acids, oleic acid, monounsaturated fatty acids, and polyunsaturated fatty acids. The word "nutraceutical" is usually used to determine food nutritional supplement that states a medical or health benefit to provide proper nourishment (Wong et al., 2015). They contain useful nutrients which are famous as a good remedy for chronic diseases (Luciano, 2014); their leaves, bark and nuts are ethnobotanically used for the cleansing of teeth (Irfan et al., 2018a, b), leaves are used for intestinal infection and eczema (Irfan et al., 2018c, d), bark is used in cleaning of teeth, toothache and in diabetes (Irfan et al., 2018e); fruits are rich in proteins used for cleaning of teeth and throat infections (Ahmad et al., 2016), bark is used in dry cough, decoction of leaves given for eczema and intestinal worms (Irfan et al., 2017a, b). The relationship between cognitive health and walnuts are inconclusive (Cahoon et al., 2021). Walnuts are an efficient nutrient that contains an important constituent in Mediterranean food and are considered as finest nutrition (Ley et al., 2014). Their green shells, bark, and kernel are mostly important for cosmetic and pharmaceutical industry (Adeel et al., 2017). They are single seeded, round stone fruits commonly used for their meat. Walnuts are ripening between September and November (Grant et al., 2011).
Generally, the nuts of Juglans regia L. are used for their nutritive features because it has vital antioxidants potential and is also considered as reserve nutrition elements (Martinez and Maestri, 2016). The seeds of walnuts are considered good for coronary and heart ailments throughout the world (Schwingshackl et al., 2017). It also has numerous nutritive aspects that indicate additional segments of the polyphenolics; therefore, it is assimilated into the nutritional food. Sanchez-Gonzalez et al. (2017) examined a number of phytochemicals in the walnuts such as fatty acids, melatonin, serotonin, and phenolics. Several phytochemicals in walnuts like hydrolyzable and condensed tannin have been studied (Figueroa et al., 2016;Irfan et al., 2019a). High oil contents as well as the protein of walnut kernels helps the fruits necessities for our nutrition. Food and agriculture organization has categorized walnuts as a deliberate candidate and phytomedicine for human food (Gandev, 2007). Generally, from fats energy can be produced from nuts that have nearby 66% and create more calories energy and considered the best nutrition (Tapsell et al., 2009). Monounsaturated fatty acids and triacylglycerols are mostly oleic acid (α-linolenic acids) and polyunsaturated fatty acids (PUFAs) (Martinez and Maestri, 2008). Linoleic acids in the oil of walnut are present in high amounts in all their genotypes. The omega-6 fatty acid is the central constituent of the walnuts which are usually known as linoleic acid. Walnut contains comparatively more quantities of the omega-3 fatty acids about 8-14% of the entire fat contented that is known as alpha-linolenic acids. Deckelbaum and Torrejon (2012) investigated those walnuts are a chief source of alphalinolenic acids that reduces inflammation and augments blood composition. Muradoglu et al. (2010) examined the From each walnut tree in specific site (1 kg) of nuts were collected. Nuts were then dehydrated and kept in plastic bags at room temperature for further chemical analysis.

Nuts samples preparation
Nuts were collected from the selected walnuts and then they were treated for research investigations. Nuts were divided into two equal amounts and shells were removed and grinded by the blinder machine and transformed to the form of powder. The powdered were then stored in paper bags for further chemical analysis.

Elemental composition
The elemental composition of nuts was determined by the wet digestion technique. By a flame photometer (Jenway PFP7) the minerals including calcium (Ca) and magnesium (Mg) were analysed (Khan et al., 2007). There investigated alkaline heavy metals such as calcium, zinc, magnesium and iron were through atomic absorption spectrometer with air/acetylene flame at the 2200-2400K (Photomultiplier tube detectors) (Hanlon et al., 1992).

Principle of the assay
Chromogens were made when there Folin and Ciocalteu's phenol chemical react with non-phenolics and phenolics reducing resources. Chromogens were sensed spectrophotometrically at 515 nm. The color progress happens of the passage in electron due to their basic pH, to decrease phosphotungstic acids/ phosphomolybdic acids for making chromogens by metals that contain fewer valence.

Preparation of Folin's reagent
Two and half grams of sodium molybdate and 10 grams of sodium tungstate were dissolved in 70 mL of distilled water and 5 mL of 85% phosphoric acid and about 10 mL concentrated HCl were combined as a solution. Then the mixture was refluxed for ten hours and 5 mL of distilled water, 15 grams of lithium sulphate, and one droplet of the bromine was added to this mixture, and reflux yet again for 15 minutes. The mixture was then cool to room temperature and distilled water was added to reach their volume up to 100 mL.

Procedure
Nuts TPC was observed by using Folin-Ciocalteu reagents by the method of Slinkard and Singleton (1977). The reagents were prepared through dilution stock solution and then added distilled water (1/10, v/v). About 5 mL of Folin-Ciocalteu's phenol chemical, 1 mL of methanolic extractions of nut, and 4.0 mL of Na 2 CO 3 (7.5%) were then mixed in the test tube. The absorbance was measured by spectrophotometer after the incubation for 1 hour at 765 nm (Shimadzu, Japan). Total phenolics contents was measured by calibration curve and showed in the mg of gallic acid equivalents/100 g of the walnuts.

Walnut conductivity
Nuts conductivity of Juglans regia L. was measured through conductivity meter. Similarly, every 15 minutes after the conductivity of nuts was measured and recorded until 60 minutes.

DPPH solution preparation
The DPPH solution of ethyl acetate was prepared at 0.1 mM amounts dissolved in 7.88 mg DPPH in ethyl acetate of 200 mL.

Procedure
Five millilitres, 0.1 mM DPPH solution was mixed in the test tube with the 56.0 µL sample in 30 mL methanol. Thirty minutes after the combination of the solution in dark and then there use UV/Visible spectrophotometer (Japan). Sample mixture absorbance was observed at 515 nm. Radical scavenging activity towards the DPPH radicals was measured by variances from the absorbance of the DPPH solution of the sample. Percentage of the RSA was calculated according to Equation 1 (Lee et al., 2007). Percent RSA was alike as percent inhibition.

Statistical analysis
Data recorded were tabulated. The experimental work was organized in a randomized design and took three replications. For comparison data were analyzed on the mean values by sigma plot version. Minitab statistical software, version 14.20 was used for data analysis. Statistical set of the social science version 23 (SPSS Inc., U.S) according to (Norusis, 2015). There Pearson correlation coefficients were also used. The variance analysis (ANOVA) was used to investigate various environmental factors mainly precipitation and temperature effect on the walnut morphological characters. After the mean values of walnut characters correlation of various climatic factors was calculated according to Equation 2 (Fulekar, 2009). Here 'y' = variable and 'r' = Correlation of coefficient

Results
The walnut biochemical parameters like radical scavenging activity, total phenolics contents, elemental composition, and conductivity of walnuts were examined at various sites of districts Swat and Dir at diverse altitudes.

Walnuts total phenolics contents
Walnuts' total phenolics contents in the investigated regions districts Swat and Dir were noted at diverse altitudinal regions.
It was decided that nuts at high altitudes which contain lower temperatures have higher total phenolic contents (Kalam and Laram) in both Districts investigated regions whereas in the low altitude which contain higher temperatures have less total phenolic contents (Rangila and Balo Rabat). There is a significant difference present in all research places (Table 1).

Conductivity of walnut
Conductivity of walnuts was investigated by the conductivity meter. Walnut's conductivity in both investigated regions districts Swat and Dir were noted at diverse altitudinal sites.
In districts Dir conductivity of walnuts was noted by conductivity meter after 15 minutes as 226. In our study we concluded that conductivity increases with an increase in the time. Walnuts at high altitudes contain lower temperatures which have higher conductivity (Kalam and Laram) in both the districts investigated regions whereas the low altitude which contains higher temperatures has low conductivity (Rangila and Balo Rabat). Significantly variance was existed in all the research places (   (Table 4). It was noted that RSA was more in walnuts at low temperature and high-altitude places viz. Malam Jaba and Laram and less at low altitude and high temperature places such as Rangila and Balo Rabat in both District Swat and Dir. Significant difference was existed in all the studied localities (Table 4).    In column similar letters values no significant difference α < 0.05. copper (Cu) and Magnesium were more in walnuts at low temperature and high altitude places viz. Malam Jaba and Laram and less at low altitude and high temperature places like Rangila and Balo Rabat whereas zinc were less at low temperature and high elevation viz. Malam Jaba and Laram and more at low altitude and high temperature places like Rangila and Balo Rabat in both district Swat and Dir. Significant differences were existed in all the studied localities (Table 5 & 6).

Discussion
The biochemicals parameters like radical scavenging activity, elemental composition, total phenolics contents and conductivity of walnuts were examined at various sites of district Swat and Dir at various elevations.

Walnuts total phenolics contents
Total phenolics contents of particular walnuts in both investigated regions of district Swat and Dir were noted at various altitudinal sites.
In district Swat more amount of TPC was investigated at Malam Jaba which was 227.8±0.4 mg/100g whereas in Dir it was noted in the Laram that were 226.2±0.5 mg/100g (Table 3). Malam Jaba and Laram annual rainfall were more that was 1660 mm and 1870 mm. The minimum and maximum temperature was less in Malam Jaba and Laram. The altitude of Malam Jaba and Laram was investigated as 2804 m and 2590.8 m respectively.
The intensity of light was maximum in that places which were recorded as 155.45±2.2 Cd and 150±1.6 Cd, whereas the temperature of soil was low detected as 19±0.35 ºC and 19±0.42 ºC . The soil was acidic having 6.90±0.16 and 6.88±0.25 pH values and have more EC was detected as 0.60±0.18 mS/cm and 0.61±0.18 mS/cm. The minimum and maximum temperature in Laram was investigated as 4.0 ºC and 24.1 ºC whereas the minimum and maximum temperature in Malam Jaba was detected as 4.2 ºC and 23.2 ºC respectively (Table 1).
In district Swat less amount of TPC was detected in Rangila that was 211.3±0.3 mg/100g whereas in Dir it was noted in Balo Rabat that was 211.2± 0.6 mg/100g (Table 5 & 6). The soil was less acidic having 6.10±0.27 and 6.11±0.15 pH values and have less electrical conductivity was recorded as 0.20±0.17 and 0.22±0.16 mS/cm. Annual rainfall of Rangila and Balo Rabat is less and 1360 mm and 1333 mm, while minimum and maximum temperature were high in Balo Rabat, was 9.8 ºC and 33.9 ºC and in Rangila that was 13.7 ºC and 33.7 ºC respectively. The altitude of Rangila and Balo Rabat was 778 m and 1062m respectively. Light intensity was lower in these places which were 93±2.4 Cd and 85.56±1.5 Cd, whereas the temperature of soil was more 28±0.22 ºC and 27±0.47 ºC (Table 1 & 3).
In both districts Swat and Dir altitude and temperature indicated a significant positive correlation with TPC whereas soil pH, precipitation, and soil Ec show a weak negative correlation. Light intensity and temperature of soil indicated a weak positive correlation with the TPC in both districts (Table 7).
It was determined that there was a great effect of temperature and altitude whereas the minor effect of light intensity, soil EC, soil pH, and rainfall on the TPC. It was also determined that walnuts of the high altitude that contain lower temperature have higher TPC (Kalam and Laram) in both districts of investigated regions whereas low altitude which contains higher temperature has less TPC (Rangila and Balo Rabat). The similar result was reported by Nerg et al. (1994) that environmental differences diverged the total phenolics contents in 3 regions of different latitudes from Finland. Muradoglu et al. (2010) investigated those nutritive contents of the walnuts vary from a cultivar to the cultivar that can be affected by their cultivators, genotypes, different ecosystems, and different soil conditions. Blomhoff et al. (2006) documented that nuts have maximum quantities of antioxidants, polyphenols, phenolic compounds and tocopherols that are situated in the nut's pellicles. Oliveira et al. (2008) documented those phenolic compounds and natural antioxidants were found in walnuts that were essentials because it reduced the risk of degenerative infections and recover their health. Ghasemi et al. (2011) documented that various climatic and geographical conditions in various areas like lowest daily temperature and highest altitude in a geographic region Abali have various bioactive compounds containing phenolics content and walnuts bioactivities. Omwamba and Hu (2009) investigated that in the highest temperature regions their total phenolics contents reduced due to their degradation.

Walnuts conductivity
Walnut's conductivity was recorded by a conductivity meter. The conductivity of walnuts in both investigated regions of district Swat and Dir were noted at various altitudinal sites.
In district Swat maximum conductivity up to 60 min after the treatment was noted in Malam Jaba which was Table 7. Correlation between climatic factors and chemical parameters of walnuts. In both districts Swat and Dir precipitations, altitude, temperature, soil Ec and light intensity indicated a weak negative correlation with the conductivity, whereas soil temperature and soil pH showed a weak positive correlation (Table 3).

Climatic Factors Districts
It was decided that there is less effect of altitude, light intensity, temperature, rainfall, soil EC and soil pH on the conductivity. It was also observed that at higher altitudes the walnut cotyledons were soft due to which electrolytes were released maximum as compared to the low altitude which contains hard cotyledons. In walnuts conductivity increases with an increase in time due to the existence of vascular opening by which their high quantity of electrolytes discharges and bulky nut size yields maximum electrical conductivity. Our results agreed with the previous study of Sivritepe et al. (2015) and Bonner (1968) who documented that the increased amount of conductivity might be due to the electrolyte that was beneath and within the walnut pericarp. Mark et al. (1990) and Matthews and Powell (2006) as well as McDonald Junior and Wilson (1980) documented that electrolyte total quantity leaked by nuts in soaking period gives us the assessment of electrical conductivity. The results also presented resemblances with the Pandey (1992) who investigated that soft nut possess a poor membrane system that results in maximum electrolyte losing and therefore indicate higher electrical conductivity.

Radical scavenging activity
The walnuts RSA in both investigated regions district Swat and Dir were noted at various altitudinal sites.
Maximum radical scavenging activity in district Swat was investigated in Malam Jaba which was 52.18±0.4% whereas in Dir it was observed in Laram which was 51.73±1.4% (Table 3). Similarly, minimum radical scavenging activity in District Swat was investigated in Rangila was 43.32±1.5% whereas in District Swat it was observed in Balo Rabat which was 43.62±1.2% (Table 3). In both districts Swat and Dir altitude and temperature indicated a positive correlation whereas light intensity, soil temperature and altitude indicated a weak positive correlation with the RSA. Soil Ec, Soil pH, precipitation and indicated weak negative correlation with the RSA (Table 4).
It was determined that the RSA was more in walnuts at low temperature and high altitudinal places such as Malam Jaba and Laram was less at low altitude and hightemperature places such as Rangila and Balo Rabat in both districts Swat and Dir.
From our results it was determined that there is much effect of temperature and altitude whereas less effect of the soil pH, light intensity, soil EC, and rainfall on the RSA. Our results showed similarities to the Ghasemi et al. (2011) that various climatic and geographical conditions in various areas such as high altitude and low temperature have more bioactive compounds and RSA in the nuts. On the other hand, RSA at higher altitudes is as low as compared to the results reported by Carvalho et al. (2010). Meanwhile Muradoglu et al. (2010) investigated those nutritive substances of nuts vary from a cultivar to another cultivar that can be affected by different ecology, cultivators, genotypes and various soil conditions. Oliveira et al. (2008) investigated that phenolics compounds and antioxidants are present in the walnuts that were essential because they reduce the risk of degenerative infections and recover our health problems. Xu et al. (2017) documented that a decrease of the antioxidant activity may take place due to the phenolic compounds degradation as they heated to a higher temperature of low altitude sites.

Walnut elemental composition
Walnuts elemental composition (Fe, Zn, Cu, Mg, and Ca) of the investigated localities of district Swat and Dir were noted at diverse altitudinal regions.
In Dir it was noted that iron (Fe), copper (Cu), magnesium (Mg) and calcium was maximum that were 0.422±0.020, 0.698±0.82, 28.7±0.234, and 17.6±0.015 mg/L respectively, whereas zinc was lower which was 0.510±0.17 mg/L in the Laram. Similarly in district Swat iron, copper, magnesium, and calcium were maximum that were 0.348±0.32, 0.543±0.65, 38.1±0.247 and14.7±0.030 mg/L respectively, whereas zinc was lower viz. 0.406±0.10 mg/L in Malam Jaba site (Table 3 & 4). In Dir it was observed that iron, copper, calcium, and magnesium were minimum that were 0.212±0.018, 0.442±0.38, 21.1±0.139 and 10.1±0.120 mg/L respectively, whereas zinc was maximum that were 0.674±0.23 mg/L in Balo Rabat. Similarly in district Swat iron, copper, magnesium and calcium was less that were 0.209±0.13, 0.592±0.06, 20.0±0.313 and 10.2±0.030 mg/L respectively, whereas zinc was more that was 0.685±0.15 mg/L in Rangila (Table 3 & 4). In both regions of district Swat and Dir the copper concentration indicated a weak negative correlation with the Soil and air temperature, light intensity whereas it presented a weak positive correlation with soil pH, altitude, precipitation and soil Ec. The zinc concentration indicated a weak negative correlation with soil Ec, soil pH, precipitation and soil temperature whereas it indicated a weak positive correlation with light intensity, altitude and temperature. The iron concentration indicated a weak negative correlation with the soil pH, temperature and soil temperature whereas it indicated a weak positive correlation with altitude, precipitation, soil Ec and light intensity. The calcium concentration indicated a weak negative correlation with light intensity and precipitation whereas it indicated a weak positive correlation with altitude, temperature, soil pH, soil Ec and soil temperature. The concentration of magnesium indicated a weak negative correlation with the soil pH, temperature, soil Ec and soil temperature whereas it indicated weak positive correlation with light intensity, altitude and precipitation (Table 4).
Coefficient correlation between all the environmental features i.e. altitude, temperature, precipitation, light intensity, soil pH, soil EC and temperature of soil with all the parameters of the elemental analysis i.e. zinc, copper, iron, magnesium and calcium were noted in (Table 3).
It was concluded that there is a small effect of light intensity, altitude and temperature, soil EC, rainfall, and soil pH on the elemental composition (Zn, Cu, Mg, Fe and Ca). It was also noted that iron, copper, magnesium, and calcium was more in walnuts at high altitude and lower temperature places viz. Malam Jaba and Sheringal and less at low altitude and high temperature places like Rangila and Balo Rabat whereas, zinc was less at low temperature and high-altitude places viz. Malam Jaba and Sheringal and more at low altitude and high temperature places like Rangila and Balo Rabat in both district Swat and Dir. Our results agreed with Cosmulescu et al. (2009) stated that minerals composition of 9 Juglans regia L. cultivars at various climatic and geographical environments instigated from Romania. Cosmulescu et al. (2009) investigated that nuts macronutrient had a rich source of vitamins, minerals like magnesium, potassium, iron, and phosphorus. Siahnouri et al. (2013) investigated that calcium, zinc, sodium and copper were present in proper percentages. Jeszka-Skowron et al. (2017) investigated macronutrients that were essential for proper body growth and development for human beings. Abdel-Aziz et al. (2016) reported that micronutrients maintain the immune system, heart health, function of bone and nervous system functions. In contrast the examined values of magnesium were nearly closer to the values investigated by (Irfan et al., 2018f;Ravai, 1992) whereas calcium, copper, zinc and iron indicated some differences.

Conclusion
In current study the effects of different climatic drivers on the chemical composition of Juglans regia L. nuts were investigated. In climatic investigations it was concluded that higher altitude regions have a low temperature of the soil, high light intensity, more precipitation, high EC have less acidic soil with more rainfall, low maximum annual temperature whereas the less altitudinal areas with less light intensity, low precipitation, more soil temperature have less acidic soil with less EC and regions with great maximum annual temperature and low rainfall. In the chemical investigations it was determined that walnuts of greater altitude have lower temperature with higher radical scavenging activity, total phenolics contents and conductivity in both districts' localities, while less altitude with maximum temperature has less total phenolics contents. The calcium (Ca), iron (Fe), magnesium (Mg), and copper (Cu) was maximum in the walnuts of low temperature and higher altitude locations and less at higher temperature and lower altitude locations while, element zinc was minimum at low temperature and high altitude sites and maximum at higher temperature and low altitude locations in both district Swat and Dir. Altitude and temperature resulted moderate correlation with total phenolic contents (TPC) and radical scavenging activity (RSA) while soil temperature, precipitation, soil Ec, soil pH and light intensity indicated a weak correlation with the walnuts chemical traits. Walnuts chemical composition was changeable that depends on their diversities, soil conditions, climatic conditions, ecology and genotypes.