Molecular Identification and Antioxidant Activity Determination among Coffee Varieties Cultivated in Nepal

Coffee is the most popular beverage containing numerous phytochemical components that have antioxidant activity capable of scavenging free radicals. Antioxidant and phenolic contents have considerable benefits for human health. The aim of this study was the molecular identification of 9 coffee samples from the Nepal Agricultural Research Council, Lalitpur, Nepal, and the determination of the antioxidant activity and total phenolic content of green and roasted coffee beans. Molecular identification was performed using ITS-specific PCR followed by sequencing and phylogenetic tree construction using the maximum parsimony method. The DPPH assay was used to determine the antioxidant activity, and the Folin–Ciocalteu (F-C) assay was used to determine the total phenolic content. All the samples belonged to the taxa Coffea arabica. The antioxidant activity in roasted beans varied from 2.49 to 4.62 AAE mg/g and from 1.4 to 3.9 AAE mg/g in green beans. The total phenolic content varied from 2.58 to 3.38 GAE mg/g and from 4.16 to 5.36 GAE mg/g for the roasted beans and green beans, respectively. The data revealed that the highest antioxidant content (4.62 AAE mg/g) was found in roasted coffee and that the highest phenolic content (5.36 GAE mg/g) was found in green coffee. The study concludes that roasting increases the antioxidant activity but decreases the phenolic content of coffee.

All cofee species originated from the intertropical forests of Africa and Madagascar [4].Today, cofee is grown worldwide in tropical and subtropical regions [5].Cofee is grown in more than 70 countries, among which Brazil is the highest producer with a production of around three million tons in 2019 [6].Te United States, Europe, and Japan are the countries with the highest cofee consumption [7].
In Nepal, cofee was introduced in 1938 A.D. from Myanmar and planted in the Gulmi district.Later in 1968 A.D., Nepal government initiated cofee development in Nepal, brought seeds from India, and distributed them to farmers.Te production of cofee is increasing with great potential to provide farm employment and income generation to farmers [8].More than 30,543 farmers from a 2646 hectare area in the midhills of Nepal, with an altitude of 700-1600 meters above sea level, are actively involved in cofee production [9].Annually, total cofee production and area of production of cofee in Nepal are increasing by 35% and 28%, respectively [10].Despite Nepal's long history of cofee farms, few taxonomy investigations have been undertaken.Te goal of this study was to identify cofee species and develop a genetic link between them.Many PCR-based molecular marker approaches were employed for genetic diversity assessments, genotype identifcation in gene bank management, molecular phylogenetic investigations, and species diagnostic procedure creation.Tese techniques include those based on random amplifed polymorphic DNA (RAPD), microsatellites or simple sequence repeats (SSRs), intersimple sequence repeats (ISSRs), amplifed fragment length polymorphism (AFLPs), and internal transcribed spacers (ITS) sequences [11].
Among various methods, DNA barcoding using internal transcribed spacers (ITSs) was more efcient in the molecular identifcation of cofee [12].Te internal transcribed spacer (ITS) region of the nuclear ribosomal cistron consists of two spacers, ITS1 and ITS2, which are located between the small subunit 18S ribosomal gene and the large subunit 26S ribosomal gene and are separated by the 5.8S gene.Characteristics such as biparental inheritance, simplicity, intragenomic homogeneity, intergenomic diversity, and minimal functional constraint have led ITS-based phylogenetic analysis to dominate the plant molecular phylogenetic approach.Along with these advantages, there is the presence of a large body of sequence data for this region; recent studies have used the ITS region to study the phylogenetic relationship among several plant genera [13,14].
Cofee is a rich source of dietary antioxidants, and this property, combined with the fact that cofee is one of the world's most popular beverages, has led to the consideration that cofee is a major contributor to dietary antioxidant intake and is regarded as a functional food [15].Cofee is a complex food matrix with numerous phytochemical components that have antioxidant activity capable of scavenging free radicals.Te most important bioactive compounds include phenolic compounds (chlorogenic acids and derivatives), methylxanthines (cafeine, theophylline, and theobromine), diterpenes (cafestol and kahweol), nicotinic acid (vitamin B3), and its precursor trigonelline, magnesium, and potassium [16].
Antioxidants are micronutrients that have gained importance in recent years because of their ability to neutralize free radicals [17].Although cofee is consumed primarily because of its pleasant favor and stimulating properties, more recent investigations have indicated potential health benefts associated with the beverage, including a reduced incidence of several chronic and degenerative diseases, such as cancer, cardiovascular disorders, diabetes, and Parkinson's disease, and reduced mortality risks [18,19].Some studies have shown that cofee components can trigger tissue antioxidant gene expression and protect against gastrointestinal oxidative stress [20].
Since the introduction of cofee in Nepal, only the socioeconomic aspect of cofee has been studied, but no major work on its genetic identifcation and diversity has been initiated.Tere are few research studies on its characterization which lay a foundation but do not give complete information.Te present work aims to evaluate the phylogenetic relationship, antioxidant content, and total phenolic content of 9 cofee samples grown in Nepal.Because cofee is the most popular beverage, the presence of antioxidant activity and phenolic contents is highly benefcial to cofee users.

Sample Collection.
Green cofee beans were obtained to access the antioxidant property, and young cofee leaves were used for molecular identifcation.Samples as shown in Table 1 were collected from the Nepal Agricultural Research Council, Lalitpur, Nepal (latitude: 27.6550983612, longitude: 85.3270125389), and stored in plastic bags with zippers.

Identifcation of the Selected Cofee Sample
(1) Sample preparation: Te leaves were cleaned and stored at −80 °C for DNA extraction.( 2) DNA extraction and sequencing: DNA was extracted from leaf samples using the Doyle and Doyle method with some optimizations [21].Te ITS2 region of genomic DNA was amplifed using primers ITSL (5′ TCGTAACAAGGTTTCCGTAGGTG 3′) and ITSR (5′ TATGCTTAAAYTCAGCGGG 3′) [22].Te PCR product was sequenced at the sequencing facility of Macrogen Inc., South Korea.Raw sequences were obtained from Macrogen.Raw sequences were assembled and trimmed using Codon Code Aligner.Te generated contig sequences were subjected to BLASTN, and the database "Standard databases (nr, etc.)" was selected.Highly similar sequences were obtained in the FASTA format for phylogenetic analysis.
(3) Maximum parsimony analysis of taxa: Te entire ITS region of cofee leaf samples was sequenced, and sequencing results were analyzed to construct a phylogenetic tree using the maximum parsimony method for the validation of the molecular identity of 9 cofee samples [23].Te bootstrap consensus tree inferred from 2000 replicates is used to represent the evolutionary history of the taxa analyzed [24].Tis analysis involved 20 nucleotide sequences.All positions with less than 95% site coverage were eliminated; i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option).Evolutionary analyses were conducted using MEGA X [25].Genetic distances among the sample sequences were used to interpret the similarities between the species.Phylogenetic analysis was used to confrm the molecular identity of cofee samples by comparing the results of nucleotide sequence alignments with those of standard sequences such as C.

Methods for Measuring Antioxidant Properties
(1) Sample preparation: Te obtained green beans were roasted at 200 °C for 20 min.Both green and roasted cofee beans were ground to powder in a standard cofee grinder.One gram of each cofee sample (both green and roasted) was resuspended in 100 mL of hot water (50-60 °C) for 3 min.Te cofee brews obtained were then fltered using Whatman Filter Paper No. 43.Finally, the cofee extracts were used for analysis, and the remaining extracts were stored at a temperature of −20 °C for future use.(2) DPPH assay: Te antioxidant activity of cofee samples was estimated according to the procedure reported by Morales and Jimenez-Perez [26] with a few modifcations.A solution of 74 mg/L DPPH in methanol was prepared fresh for the assay.20 μL aliquot of the sample was added to 1 mL of DPPH, shaken, and incubated for 30 min in the darkroom at room temperature.Absorption was measured at 520 nm using a UV-Vis spectrophotometer.Te reaction mixture containing control (1 mL methanol) and reference standard (1 mL DPPH) was also measured.Te measurement was compared with a calibration curve prepared with ascorbic acid at various concentrations (0.01-0.4 mg/mL) [27].Te results were expressed in AAE mg/g using the standard curve equation: where y is the DPPH absorbance at 520 nm and x is the ascorbic acid concentration in diferent samples expressed in mg/ml, which is then expressed as AAE mg/g of dry cofee powder.
(3) Assessment of total phenolic content (TPC) of cofee grounds: Te TPC of cofee grounds was determined by the spectrophotometric method using Folin-Ciocalteu (F-C) reagent [28].A 20 μL sample was added to a tube containing 1 mL deionized water, followed by the addition of 100 μL Folin-Ciocalteu (1 : 10) reagent.Te reaction mixture was incubated for 3 min at room temperature.Ten, 280 μL of 25% w/v sodium carbonate solution and 600 μL deionized water were added to the reaction mixture and mixed properly.After 1 h of incubation at room temperature in the dark, absorbance was measured at 765 nm using a UV-Vis spectrophotometer against the blank solution (F-C reagent and water only).Te measurement was compared to a calibration curve prepared with gallic acid solution at a concentration range (0.0005-0.5), and the total phenolic content was expressed as GAE mg/g using the standard curve equation: where y is the absorbance at 765 nm and x is the total phenolic content in the diferent samples expressed in mg/mL, which is then expressed as GAE mg/g of dry cofee powder.

Statistical Analysis.
Data analysis was performed using OriginPro 9.0 and Microsoft Excel.ANOVA was performed for quantitative data, and the Tukey test was used to compare the means at a 95% confdence interval.

Result and Discussion
Te entire ITS region of cofee leaf samples was sequenced, and sequencing results were analyzed to construct a phylogenetic tree using the maximum parsimony method to validate the molecular identity of nine cofee samples.Genetic distances among the sample sequences were used to interpret the similarities between the species.Phylogenetic analysis was used to confrm the molecular identity of cofee samples by comparing the results of nucleotide sequence alignments with those of standard sequences such as C. arabica, C. canephora, and C. congensis.Te Scientifc World Journal Te pairwise nucleotide-sequence divergence (Jukes-Cantor model) among the cofee taxa ranged from 0% to 0.9% within diferent taxa of C. arabica, as shown in Table 2.A divergence of 0.9% was detected between Syangja_Special and NARC-C1.NARC-C4 and NARC-C1 were 0.75% divergent from each other.NARC-C4, NARC-C3, NARC-C2, and Gulmi_Local were detected with 0.6% divergence from Syangja_Special.All samples, except Syangja_Special, were detected with no divergence with NARC-C5.Tese divergences were attributable to deletion and insertion events, and gaps were introduced to align the sequences that appeared particularly important within the diferent cofee species [4].
Maximum parsimony (MP) analyses resulted in a single, fully resolved tree with a length of 242, a consistency index of 1.0000, and a retention index of 1.0000.Te maximum parsimony tree (Figure 1) shows that all the samples in this study were C. arabica.Te most efective method for DNA barcoding of plants involves the integration of both coding and noncoding genetic markers.Most of the research on barcoding uses conserved regions such as rbcL and matK regions which show great variability.Regardless, ITS is considered a standard barcode for most of the plant and some algal species [30].
As the molecular identifcation of Nepali cofee genotypes was unknown, the genotypes were cultivated solely on verbal information.Very few studies have been undertaken in Nepal for the identifcation of cofee species.Most of the studies were based on socioeconomic aspects, and there was no sufcient information regarding its identifcation and diversifcation.Previously, a phylogenetic analysis of cofee samples from Gulmi (Cofee Development Center) was undertaken, which showed that all the samples were Cofea arabica [12].Tese studies provide valuable information regarding the status of cofee genotypes for farming and varietal improvement.It acts as a starter for identifcation studies of cofee found in Nepal, with a prominent aspect of researching for better quality output for the farmers of Nepal.
Homogenization mechanisms linked with concerted evolution are thought to maintain overall sequence homogeneity among members of a gene family, such as nuclear rDNA [31].As a result, rDNA repeats are usually very similar within individuals and species, although diferences may accumulate between species, as we observed in pairwise comparison [32].Te observed defciency in the homogenization mechanisms may be related to the long-life cycles of cofee trees, in the same manner, that nucleotide substitution rates have been reported to be related to the length of the reproductive cycle [5,33].Moreover, it is most likely that spontaneous interspecifc hybridization occurs between taxa and is involved in speciation.Given their important role in posttranslational processing, ITS regions are considered to be quite conserved [34].However, ITS phylogeny was successful in distinguishing the cofee species in our investigation.Further use of other phylogeny techniques must be carried out to identify and validate the species level of genotypes.Nevertheless, this discovery lends credibility to the use of ITS as a phylogenetic reconstruction technique for intraspecies diferentiation in cofee species.
Te antioxidant activity expressed as milligrams of ascorbic acid equivalent antioxidant capacity per gram cofee extract (AAE mg/g) was determined for 9 cofee samples (green and roasted).Te antioxidant activity in roasted beans varied from 2.49 to 4.62 AAE mg/g and from 1.4 to 3.9 AAE mg/g in green beans as shown in Figure 2(a).Te concentration of ascorbic acid equivalent compounds was highest in a roasted sample (Gulmi_Local) and lowest in a green cofee bean (NARC-C5) at a confdence interval of p < 0.05.Te average value ranged from 2.5 to 4.0 AAE mg/g for green and roasted cofee beans as shown in Figure 2(b).A similar study found that the Trolox equivalent antioxidant capacity varied from 0.88 to 1.29 TEAC mg/g and from 0.08 to 1.83 TEAC mg/g in green and roasted cofee samples, respectively [35].
Te results suggest that green beans possess antioxidant activity, but the activity is signifcantly higher in roasted beans.Green cofee beans are rich in bioactive compounds such as cafeine, trigonelline, chlorogenic acids, tocopherols (α, β, c), and diterpenes (mainly kahweol and cafestol) with antioxidant properties [36].Dark-roasted cofee has a signifcantly low antioxidant activity, whereas lightroasted cofee has the highest antioxidant activity [37,38].A study showing the efect of roasting conditions on the antioxidant activity of Colombian cofee illustrates that roasted beans exhibit a signifcantly higher antioxidant activity than unroasted beans, which may be due to the Maillard reaction and the release of bound polyphenols from plant cells [39].Because antioxidant compounds provide health benefts, cofee as a popular beverage will be of great interest to those trying to increase their intake of these nutrients.
Among the nine cofee samples, eight roasted samples exhibited an increased antioxidant activity compared with their respective green beans, whereas in sample Syang-ja_Special, the opposite was observed, as shown in Figure 3, and specifcally, in varieties Gulmi_Local, Kshetradeep, NARC-C2, NARC-C3, and NARC-C5, the roasted beans exhibited a signifcant increase in antioxidant activity compared with the green beans by 30.13%, 20.45%, 24.76%, and 36.74%,respectively.However, in Syangja_Special, the antioxidant activity decreased by 7.69% after roasting.In a similar study, roasted beans showed a signifcantly (p < 0.01) higher antioxidant activity than green beans by 24.1%-27.9%[28].High temperatures employed during roasting are well recognized to have a signifcant impact on the chemical makeup of cofee beans [33,34].During roasting, some substances, such as chlorogenic acids, are broken down, whereas other compounds, such as melanoidins, are composed [10,35].
Total phenolic content (TPC) was expressed as milligrams of gallic acid equivalent compounds per gram of cofee extract (GAE mg/g).Phenolic compounds were found to be signifcantly higher in unroasted samples than in roasted samples.Figure 4(a) illustrates that the highest TPC was found in a green sample (Sankhuwasabha_Red) and the lowest in a roasted sample (NARC-C2) at a confdence interval of p < 0.05.TPC varied from 2.58 to 3.38 mg GAE/g and from 4.16 to 5.36 mg GAE/g for the roasted beans and Te Scientifc World Journal green beans, respectively.Te average value ranged from 2.93 to 4.65 GAE mg/g as shown in Figure 4(b).Our fndings were remarkably comparable to those of a study that found that the extractable fractions of green and roasted cofee beans had TPCs that ranged from 114.71 to 172.49 and 92.03 to 134.70 mg of GAE per 100 grams, respectively [40].All green cofee samples possessed a higher number of phenolic compounds than roasted beans.Tis is because the total phenolic content and antioxidant activity of cofee beans are signifcantly infuenced by the roasting levels and geographic origin.During roasting, phenolic compounds are partially degraded and/or bound to polymer structures depending on roasting conditions [29].Te low concentration of polyphenols obtained in the present study is in agreement with those presented in the relevant literature, even though, depending on the variety, large variations have been detected [36,41,42].Te lower concentration of polyphenol could be explained by the fact that phenolic compounds are often more soluble in alcohol extracts than water, which was used in this study [35].
Te results showed that the samples were C. arabica.According to various studies, arabica cofee has a higher antioxidant activity and total phenolic content [43,44].From this perspective, considering the high antioxidant activity and phenolic content, consumption of these cofees has potential health benefts.Te number of base substitutions per site between sequences is shown.Analyses were conducted using the Jukes-Cantor model [29].Te rate variation among sites was modeled using a gamma distribution (shape parameter � 1).Te Scientifc World Journal

Conclusion
All of the samples in our investigation were Cofea arabica, with little or no variation within the species.In this study, the concentration of compounds exhibiting antioxidant activity in roasted cofee beans was greater than that in green beans.Te highest concentration of ascorbic acid equivalent compounds was found in Gulmi_Local among the roasted beans and Syangja_Special among the green beans.Green cofee beans possessed a higher number of phenolic compounds than roasted beans.Te total phenolic content was found to be highest in NARC-C4 among the roasted samples and Sankhuwasabha_Red among the green samples.Tus, roasting afects the chemical makeup of cofee beans by generating and breaking down compounds and afecting the antioxidant activity and phenolic contents of cofee beans.
Tese fndings help us understand the genetic relationships between numerous cofee varieties in Nepal.Based on these fndings, further studies might examine the genetic diversity of cofee varieties in Nepal and perhaps enhance the region's cofee-growing methods.

1 OP159452Figure 1 :Figure 2 :Figure 3 :
Figure 1: Maximum parsimony tree generated from internal transcribed spacer (ITS) sequence data.Branches corresponding to partitions reproduced in less than 50% of bootstrap replicates are collapsed.Te percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to the branches.

Figure 4 :
Figure 4: (a) Graph showing the comparison of gallic acid equivalent phenolic compounds in cofee samples.* Te gallic acid equivalent phenolic compound (roasted samples) is signifcantly lower than the mean value of NARC-C4 at α � 0.05.* * Te gallic acid equivalent phenolic compound (green samples) is signifcantly lower than the mean value of Sankhuwasabha_Red at α � 0.05.(b) Graph expressed as the mean concentration of phenolic compounds of green and roasted cofee beans expressed in GAE mg/g.

Table 1 :
Descriptions of 9 cofee samples used in this study.� green; R � red.Data received from the Nepal Agriculture Research Center, NARC. G

Table 2 :
Pairwise comparisons of ITS2 sequences obtained from the nine cofee trees listed in Table1.