Polyphenol and flavonoid content in major Teff [Eragrostis tef (Zuccagni) Trotter] varieties in Amhara Region, Ethiopia.

Teff [Eragrostis tef (Zuccagni) Trotter] is a small-sized cereal grain and an indigenous crop in Ethiopia. The Amhara region is one of the major teff producers regions in the country. However, information on the phenolic content of the region's teff varieties is limited. Seventy-two teff samples were collected from three administrative zones (West Gojjam zone, Awi zone, and East Gojjam zone) of the Amhara region of Ethiopia. The samples' total polyphenol and flavonoid contents were determined using colorimetric methods. The total flavonoid contents expressed as catechin equivalent, CE (i.e., under alkaline conditions) and quercetin equivalent, Q.E (i.e., under the methanolic solution of AlCl3) were found to be in the range of 7.66 ± 0.60-57.36 ± 3.87 mg C.E and 15.45 ± 0.15-113.12 ± 3.09 mg Q.E per 100 g of teff samples, respectively. The corresponding total polyphenol content (TPC), described as gallic acid equivalent (G.A.E.), was in the range of 46.21 ± 1.20-133.32 ± 5.44 mg G.A.E. The results showed that the mean TPC value of the teff samples from the West Gojjam zone was enriched with polyphenol than samples from the Awi zone and East Gojjam. Furthermore, it was noted that the mean TPC and TFC values did not vary significantly between samples of the East Gojjam and Awi zone (p > 0.05). In contrast, a significant difference in mean TPC and TFC-Q.E were noted between the sampling zone of East Gojjam and West Gojjam and between West Gojjam and Awi zones (p < 0.05). These significant variations in TPC and TFC might be due to observable variations in the agroecological zones and the genetic-make-up of the samples. Person correlation indicated a significant positive correlation matrix between the three variables (p = 0.01). The teff samples were trying to be classified based on their geographical origin using hierarchical cluster analysis (HCA) and biplots. Accordingly, the variance explained by component 1 (PC1) is 67.2%, and the variance explained by component 2 (PC2) is 20.0%.

Introduction

Teff [Eragrostis tef (Zuccagni) Trotter] is an ancient small-sized tropical cereal that has its center of origin and has diversified in the Ethiopian highlands, where it is believed to have been domesticated [1]. It is used as a cereal crop in the Poaceae or Gramineae family with small grains and originated in Ethiopia between 4000 and 1000 BC [2]. In other countries, like Australia, South Africa, and the United States, it is used as a forage crop for animal feed and as a thickener for soups, stews, and gravies [3].

Teff has a naturally higher nutritional value and is rich in essential amino acids and minerals such as Fe, Zn, Mg, and Ca than cereals such as maize and pearl millet. On the other hand, a comparable level of protein and carbohydrates is reported in teff grains compared to the most common cereal grains such as wheat, barley, sorghum, and pearl millet, and it is a gluten-free cereal grain [4,5]. As many people are diagnosed with celiac disease and other forms of gluten sensitivity, the demand for gluten-free meals is increasing. As a result, the nutrient profile of teff grain suggests that it has a lot of potential for application in foods and beverages around the world [2,6].

Teff is a staple food in Ethiopia, and the flour of teff is mainly used for making “injera” which is the favourite national dish. The “injera” prepared from teff has a pleasing odour, flavor, texture, and quality [7,8]. Teff is a chasmogamous C4 annual cereal with a fibrous root system and usually upright stems, while some cultivars have to bend or elbow varieties [9,10].

Teff grain is tiny and occurs in various colors, ranging from pale white to ivory white, light brown to dark brown, and reddish-brown purple. This color determines grade and market price [10,11].

Teff is adapted to growing environmental conditions such as drought and waterlogging. It can thrive in some harsh climate conditions when major crops might fail [7]. It is also less vulnerable to weevils and other pests during storage than other cereal grains [5]. It has the greatest area under cereal cultivation and is Ethiopia’s third-largest grain producer (after maize and wheat) [12,13].

Different teff varieties are on the market. In terms of color, the most common teff varieties are white, brown, and red. Brown teff is also known as mixed-colored teff. It contains natural mixed white and red-colored teff grains. The report indicated significant variation in minerals and other nutritional constituents between the three colored variants [14,15]. The white variety of teff is smaller in size. Red teff has better mineral and essential amino acid compositions and slightly higher carbohydrate, fibre, and energy content [4,16]. White teff is the most widely used teff among customers and is mainly grown in Ethiopia`s highlands, under the most challenging conditions. It is the most expensive type of teff [7].

Whole-grain cereal intake has been provided to effectively reduce the risk of cardiovascular disease, type 2 diabetes, ischemic stroke, obesity, and cancer [17,18]. Phenolic, Fiber, and other bioactive components are the quickest to attribute to these therapeutic benefits [19-21].

Polyphenols are secondary plant metabolites that adequately defend plants from diseases and ultraviolet radiation [22]. In addition, polyphenolic compounds also protect cell constituents from oxidative damage, lowering the risk of oxidative stress-related illnesses [23,24].

Polyphenols such as flavonoids and phenolic acids are found in cereal grains in different proportions. Their concentrations are significantly affected by the genetic make-up of the crop and environmental conditions. These phytochemicals are responsible for the consumer’s health benefits [25,26].

As information obtained from Amhara Region’s Bureau of Agriculture, about 42 improved varieties of teff have been registered in the country, but only a few are widely cultivated in the region. Among the varieties of teff, Kuncho teff (DZ-CR-387) and Tseday teff (DZ-CR-37) are white by color and widely cultivated in the northwestern part of the Amhara region. Kuncho teff is cultivated in East Gojjam and West Gojjam zones, while Tseday teff is mainly grown in the Awi zone. In addition, red teff and brown teff varieties are also cultivated in the region, but consumers prefer these colored varieties less. Brown (mixed) teff is a variety of teff that is locally called “sergegna” and naturally exists as a mixture of white and red-colored teff varieties. Kuncho and Tseday teff varieties have a high market price and are most preferred by consumers. But the same variety of teff produced in different zones and districts have different qualities and market prices, hence having consumer preferences one over the other. Since the Amhara region is one of the most teff-producing areas of the country, there is no report on the level of total phenolics and flavonoids in different teff varieties cultivated in this region.

Thus, this study was intended to quantify the level of total phenolic and total flavonoid contents in white teff varieties (Kuncho teff (DZ-CR-387) and Tseday teff (DZ-CR-37)), Brown (Mixed) teff, and, Red teff varieties collected from 21 sub-districts of three zones (East Gojjam, West Gojjam, and Awi). Furthermore, the study aimed to see the variation of total flavonoid content with the two procedures and perform some classification models based on their geographical origins using the total phenolic and flavonoid contents.

Materials and methods

Sample collection and Pre-treatment

Seventy-two (72) teff samples were directly collected from model farmers in the three administrative zones of the Northwestern part of the Amhara region of Ethiopia. Based on the information obtained from the Amhara region’s agriculture bureau, the Northwestern Amhara region is one of the country’s most significant teff-producing areas.

Four varieties of teff such as Kuncho (white teff), Tseday (white teff), Brown (mixed), and Red teff were collected from model farmers in January 2020. About 500 g of each teff variety sample was collected and stored in an airtight plastic bag. From the East Gojjam zone, three districts (Aneded, Shebelberenta, and, Enemay) and nine sub-districts (Jamadidik, Gudalema, Adisge yegewera, Yeidwuha town, Weregona akababiw, Gedaiyesus,Weyira gurazam, Huletamiba dibisa, and Enekorna adis amba) were selected. From the West Gojjam zone, two districts (Goinjkolela and Adet/Yilmanadensa) and seven sub-districts (Zanat, Goinj, Kore tenkere, Mentadeber, Kilelet, Adet zuria, and Mosbo) were selected. Five sub-districts (Gisayita, Gangana, Ahiti, Zigem town, and, Gudarjawwi) were chosen from the Zigem district of the Awi zone. Fig 1 shows the map of sampling sub-districts and zones. After samples were transferred to the laboratory, any contamination like dust particles, soil, and husk were separated by sieving with a 0.5mm mesh size sieve and milled with an electronic grinder.

Fig 1

Map of Amhara regional state administrative zones and sub-districts showing the sampling sites of teff samples.

Chemicals, reagents, and instruments

Folin-Ciocalteu reagent, gallic acid, Catechin, Quercetin and Methanol (from Sigma–Aldrich Chemie, Steinheim, Germany), AlCl3 (from Fluka Goldie, Mumbai, India), Na2CO3 and NaOH (from BLULUX Laboratories, India), and NaNO2 (from Guangzhou Jinhuada Chemical reagent Co., Ltd, China) were obtained. UV-VIS spectrophotometer (Cary60, Agilent Technologies, USA) was used to measure the prepared standards and sample solutions absorbance. A quartz cuvette with a 1 cm path length was used as a sample holder., DAIHAN Scientific ultrasonic cleaner (WUC-D10H, Korea) was used for sample extraction, a Centrifuge (800–1, Japan) was used to centrifuge the extracts, and deionized water was used throughout the experiment.

Optimization of the extraction method

Teff flour (1.0 g) was taken in a 50 mL nylon centrifuge tube and soaked for 2 hours in 7 mL of 80:20 methanol-distilled water. The wetted sample was extracted under an ultrasonic water bath [27] for 30 min, 45 min, and 60 min at a fixed temperature (35°C). The mixture was centrifuged for 15 min at 3500 rpm, where the supernatant was decanted carefully into a second centrifuge tube. The residue was re-extracted with the same solvent ratio and volume for the second time. The supernatants from the two extracts were combined and filtered through a nylon membrane syringe filter with a 0.45μm pore filter. It was noted that 45 min extraction time provided higher total phenolic content and was used as an optimum extraction time for all the samples.

Determination of total phenolic content by Folin–Ciocalteu assay

The Folin–Ciocalteu assay was performed to estimate the total phenolic content (TPC) in the extracts following the procedure developed by Singleton et al. [28] and modified by Atlabachew et al. [29]. Briefly, 0.6 mL of the extract was mixed with 1 mL of Folin–Ciocalteu reagent and 4 mL of distilled water. After 5 min, 4 mL of 7.5% sodium carbonate solution was added and kept in the dark for 2 hours at ambient temperature before taking the absorbance at 765 nm. Similarly, a calibration curve was constructed using gallic acid standard over a concentration range of 1 mg/L to 100 mg/L and reported as mg gallic acid equivalent per 100 g of teff sample (mg G.A.E./100 g). Each measurement was conducted in triplicate.

Determination of total flavonoid content as catechin equivalent (TFC-C.E)

In this study, the catechin-like flavonoids were estimated as catechin equivalent following the method reported by Pekal and Pyrzynska [27] using AlCl3, NaNO2, and NaOH solutions. Briefly, 2 mL of each catechin standard solution or sample solution was taken and treated with 0.6mL of 5%NaNO2. After 6 min, 0.6 mL of 10% AlCl3 solution was added, shaken, and left to stand for 6 min. Finally, 4mL of 4% NaOH solution was diluted to 8 mL with 80% aqueous methanol. After 15 min, absorbance was taken at 510 nm using a UV-VIS spectrophotometer [30]. The calibration curve was constructed over a concentration range of 1 mg/L to 50 mg/L of catechin, and results were expressed as mg catechin equivalent per 100 g of sample (mg C.E./100g). Each sample was analysed in triplicate.

Determination of total flavonoid content as quercetin equivalent (TFC- Q.E)

Quercetin-like flavonoids were determined using a methanolic solution of aluminum chloride. Exactly 1mL of sample solution was taken and treated with 5 mL of 2% AlCl3 dissolved in methanol and was diluted to 10 mL with distilled water and left to stand for 60 min until the yellow color was developed. Finally, the absorbance of the resulting solution was measured at 425 nm [31,32]. The calibration curve was constructed over 0.1–80 mg/L of quercetin, and results were expressed as mg quercetin equivalent per 100 g of sample (mg Q.E./100g). Each sample was analyzed in triplicate.

Statistical data analysis

One-way variance (ANOVA) was used to evaluate the mean values of total phenolic content (TPC), total flavonoid content as quercetin equivalent (TFC-Q.E), and total flavonoid content as catechin equivalent (TFC-C.E) at 95% confidence level using Stata .14, Origin 8.0, IBM SPSS statistics 23, and Excel statistical soft-wares. The ANOVA analysis was used to know whether the variation of the mean of polyphenol content and flavonoid content was significant or not between the sampling zones. The observed datasets were chemometrically handled using Principal component analysis (PCA) to see the loading eigenvectors and the extent of variations. Principal Component Analysis (PCA) is a technique used for data reduction and improves the interpretability of data with low information loss.

Results and discussion

The UV-Vis absorbance versus wavelength spectra and calibration curves of gallic acid, catechin, and quercetin standards used are depicted in Figs 2A and 2B, 3A and 3B and 4A and 4B, respectively. A good linear response over a concentration ratio of 1.00–100.00 mg/L, 1.00–50.00 mg/L, and 0.10–80.00 mg/L, respectively, for gallic acid, catechin, and quercetin, respectively, was noted.

Fig 2

UV-Vis spectra of Gallic acid standard (a) and its calibration curve (b).

Fig 3

UV-Vis spectra of Catechin standard (a) and its calibration curve (b).

Fig 4

UV-Vis spectra of Quercetin standard (a) and its calibration curve (b).

The obtained results for 72 teff samples collected from the model farmers of different geographical origins are reported in Table 1.

Table 1

Concentration (mean±standard deviation) of TPC (mg G.A.E./100g), TFC-C.E (mg C.E./100g), and TFC-Q.E (mg Q.E./100 g) of teff samples from 21 sub-districts of Amhara region.

zones	Districts	Sub-districts	Types of Teff	Concentration of TPC in mg G.A.E./100g	Concentration of flavonoid in mg C.E. /100g	Concentration of flavonoid in mg Q.E. /100g
EastGojjam	Aneded	Jamadidik	Kuncho	70.17±2.42	10.36±0.80	30.29±0.30
			Kuncho	78.92±6.10	19.06±0.43	36.86±0.77
			Kuncho	53.34±2.78	9.89±0.11	28.91±1.16
		Gudalema	Kuncho	70.30±4.32	20.01±0.73	30.83±1.78
			Kuncho	62.24±0.45	16.47±0.73	25.37±1.82
			Kuncho	64.21±2.12	47.91±4.24	58.74±4.12
		Adisge yegewera	Kuncho	69.43±3.17	10.73±0.59	24.59±1.23
			Kuncho	78.50±1.62	11.78±0.05	31.95±2.02
			Kuncho	70.90±0.43	15.83±0.73	31.38±0.48
	Shebelberenta	Gedaiyesus	Kuncho	61.30±1.99	13.12±0.65	25.95±1.17
			Kuncho	85.57±4.81	22.73±0.15	29.83±0.21
			Kuncho	78.49±4.66	13.73±0.37	27.22±0.89
			Kuncho	46.21±1.54	7.66±0.60	15.45±0.15
			Kuncho	87.61±5.05	14.97±0.14	23.77±1.05
			Kuncho	82.55±6.57	15.87±0.33	24.94±1.88
		Yeidwuhatown	Kuncho	64.66±0.68	16.80±0.09	30.81±0.32
			Kuncho	58.06±3.65	16.36±1.17	23.88±1.54
			Kuncho	92.20±3.06	15.31±0.45	22.62±1.39
		Weregona akababiw	Kuncho	75.15±2.48	54.30±0.73	60.53±5.17
			Kuncho	64.49±4.68	16.09±0.78	29.96±0.42
			Kuncho	66.73±2.40	14.11±0.45	20.60±1.35
	Enamey	Weyira gurazem	Kuncho	79.51±4.47	32.57±0.89	27.38±0.02
			Kuncho	68.00±1.46	22.96±0.81	29.62±1.46
			Kuncho	60.12±3.00	17.86±0.35	27.16±1.47
		Huletamiba dibisa	Kuncho	70.38±0.42	22.27±0.28	29.64±1.35
			Kuncho	77.86±2.57	26.37±1.40	29.56±1.52
			Kuncho	72.33±3.07	24.66±0.04	29.48±1.05
		Enekorna Adisamba	Kuncho	73.81±0.01	35.13±1.71	36.57±1.21
			Kuncho	77.96±5.02	32.82±0.95	33.93±1.51
			Kuncho	79.98±1.90	36.62±2.04	33.98±1.52
WestGojjam	Gonjikolela	Zanat	Kuncho	77.14±1.09	17.10±0.33	36.64±2.66
			Kuncho	77.11±2.55	19.12±0.77	31.23±2.60
			Kuncho	86.34±2.84	17.22±0.38	46.38±0.60
		Gonj	Kuncho	122.63±2.68	26.34±0.33	50.43±1.14
			Kuncho	118.52±1.31	35.72±1.58	33.08±1.32
			Kuncho	66.87±1.77	19.02±0.71	42.74±1.20
		Kore tenkere	Kuncho	51.07±2.26	17.34±0.19	20.83±1.68
			Kuncho	116.89±3.03	24.42±0.18	60.00±4.28
			Kuncho	113.29±8.86	31.62±0.82	69.52±2.05
		Mentadeber	Kuncho	123.00±8.24	28.35±0.05	81.23±3.77
			Kuncho	98.56±5.27	19.78±0.61	38.39±3.41
			Kuncho	105.26±1.53	18.78±0.50	79.30±2.22
	Adet/ Yilmanadensa	kilelet	Kuncho	110.10±4.31	26.36±1.67	40.57±1.67
			Kuncho	108.91±2.67	19.15±0.90	57.98±4.34
			Kuncho	128.51±1.20	24.14±1.58	58.51±1.27
		Adet zuria	Kuncho	92.02±2.03	25.37±0.48	87.74±1.98
			Kuncho	89.66±2.05	30.45±1.03	78.52±2.05
		Mosbo	Kuncho	46.60±1.20	15.94±0.93	37.19±2.77
			Kuncho	78.12±2.81	28.93±2.50	48.20±4.11
			Kuncho	90.95±7.86	22.59±0.46	53.28±0.40
			Kuncho	94.99±2.10	19.50±0.22	74.90±3.48
Awi	Zigam	Gisayita	Brown (mixed)	104.44±3.42	35.58±1.70	48.31±1.90
			Brown (mixed)	133.32±5.44	57.36±3.87	113.12±3.09
			Brown (mixed)	117.44±2.50	34.04±2.52	42.66±1.55
			Tseday	72.94±1.63	18.03±0.49	60.39±2.27
			Tseday	70.54±0.64	17.68±0.63	36.61±2.52
			Tseday	98.58±4.16	23.48±0.24	30.74±2.36
			Red	86.32±0.25	26.80±1.96	80.18±0.09
			Red	80.25±2.92	50.93±2.90	73.80±2.97
			Red	80.24±0.91	48.25±3.30	73.64±4.94
		Zigam town	Tseday	68.52±1.14	18.63±1.65	37.49±0.86
			Tseday	75.56±3.21	17.42±0.29	39.66±3.02
			Tseday	69.80±3.62	19.29±0.01	38.32±3.65
		Ahiti	Tseday	75.44±4.08	16.29±0.46	44.10±1.90
			Tseday	68.86±0.32	21.38±0.36	33.48±2.02
			Tseday	75.55±2.73	20.54±0.59	57.22±0.86
		Gagasta	Tseday	88.80±3.58	21.50±1.78	37.13±0.43
			Tseday	58.92±1.73	11.33±0.58	35.39±1.34
			Tseday	53.77±2.21	17.78±0.68	30.38±1.68
		GudarJawi	Tseday	66.29±0.68	15.88±0.54	23.05±1.05
			Tseday	62.22±2.68	18.18±0.88	31.52±0.73
			Tseday	64.01±0.52	20.04±0.72	34.18±2.51

The highest TPC (133.32 mg G.A.E./100g) was noted in the sample from the Gisayita sub-district of the Awi zone. At the same time, the minimum concentration (46.21 mg G.A.E./100 g) was obtained in the sample from the Gedaiyesus sub-district of the East Gojjam zone. As indicated in Table 1, the total phenolic content in teff samples from the Awi zone ranged between 53.77–133.32 mg G.A.E./100g of teff samples. On the other hand, the phenolic content recorded in samples from the East Gojjam and West Gojjam zones ranged between 46.21–92.20 mg G.A.E/100g and 46.60–128.51 mg G.A.E./100g, respectively.

The brown or mixed teff variety from Awi zone contained a higher total phenolic content than the white teff (Tseday variety) collected from the Awi zone. The mean TPC values were compared between the two white teff varieties (Kuncho and Tseday) collected from the three administrative zones. It was noted that the Kuncho teff variety collected from the West Gojjam Zone contained a higher mean TPC value than the same Kuncho variety collected from the East Gojjam zone and the other variety (Tsedey) from the Awi zone. The maximum TPC was about 2.85 times as high as its minimum TPC value for each sampling zone.This indicates high variability in TPC among samples collected from different sampling zones.

The TPC results of the present study were compared with reported data. An unknown variety of samples collected from the Netherlands and Italy were found to have a TPC value of 175.65 mg G.A.E./100 g and 173.20 mg GAE/100g, respectively [33,34]. Similarly, brown teff from Bolivia and USA contained 186 mg G.A.E./100g and 219 mg G.A.E./100g, respectively. Whereas 142 mg G.A.E./100g and 141 mg G.A.E./100g of TPC were noted in white teff samples from these two countries with the same order [25]. An unknown variety of teff collected from a marketplace in Addis Ababa, Ethiopia, reported a TPC of 123 mgG.A.E./100g [35]. All these reported data were relatively higher than the mean TPC value of the present study (71–95 mg G.A.E./100g of teff samples). The possible reason for such variation might be differences in genetic resources of the raw material, agronomic practice, and environmental conditions of the sampling regions. On the other hand, 263 mgG.A.E/100g and 448 mgG.A.E /100g, respectively, for white (Tseday type) and brown teff varieties collected from the Tigray region of Ethiopia were reported by [14]. In this study, the total flavonoids in the 72 teff samples were determined following two approaches. Reports indicate that not all flavonoids react with AlCl3 in the presence of sodium nitrite and sodium hydroxide. Instead, flavonols and flavones classes of flavonoids such as quercetin, quercetrin, galangin, and rutin, are selectively determined at 410–430 nm by reacting the sample solutions with AlCl3 in acidic solution acetate buffer or methanolic solutions. In contrast, flavan-3-ols (catechins and others) could selectively react with AlCl3 solution in sodium nitrite and alkaline conditions, and measurement can be done at 510 nm [30]. Thus, flavonoid data obtained with only one of the approaches could not reflect the total flavonoid content in a given sample. The total flavonoid content as quercetin equivalent (TFC-Q.E) ranged from 15.45–113.12 mg Q.E./100g of teff sample, as indicated in Table 1. The lowest and highest TFC-Q.E were found in samples from the East Gojjam zone (15.45 mg Q.E./100g) and the Awi zone (113 mg Q.E./100g). Generally, teff samples from the Awi zone had the highest TFC compared to the other two zones. This comparative difference might be due to the varietal type effect and other environmental conditions. The total flavonoid content expressed as catechin equivalent ranged from 7.66–57.36 mg C.E./100g. The lowest concentration of TFC-C.E was noted in sample from the East Gojjam zone, and the highest was recorded in the teff sample analyzed from the Awi zone.

In this study, the amount of flavonoid as quercetin equivalent was higher than the total flavonoid content as determined with catechin equivalent. On average, the TFC-Q.E content in the teff samples is 1.5 times higher than the TFC done with catechin equivalents. Similar to the TPC value, the brown (mixed) teff samples from the Awi zone had higher flavonoid contents (Table 1) than the white variety from this sampling zone. Comparing the mean value of TFC between the three sampling zones, samples from the west Gojjam zone had a mean TFC-Q.E value higher than samples from East Gojjam and Awi zones.

Looking at the reported data by [25] for teff samples collected from USA and Bolivia, the TFC ranged between 62–67 mg rutin equivalent/100g in white teff and 106–116 mg rutin equivalent/100g) of brown teff samples. Similarly, a relatively higher concentration of total soluble TFC (41–63 mg C.E /100g of sample) was reported in white and brown varieties of teff collected from the Tigray region of Ethiopia [14].

The ANOVA test indicated in Table 2 confirmed significant variations (p < 0.05) in the total phenolic content among the 72 teff samples. The flavonoid content assessed by catechin equivalent (TFC-C.E) shows no significant difference (p > 0.05) between the samples taken from the three administrative zones. As indicated in Fig 5, the value of TPC and TFC-Q.E were found to be the highest in West Gojjam teff samples, followed by the Awi zone. TPC- G.A.E were highest in samples collected from the West Gojjam zone, followed by Awi and East Gojjam administrative zones. Looking at the error bars in Fig 5, the TPC, TFC-Q.E, and TFC-C.E in teff samples from the Awi zone and West Gojjam had wide variability within the sampling zones.

Table 2

Analysis of variance (ANOVA) among 72 teff samples from three among three administrative zones.

ANOVA test
		Sum of Squares	Degree of freedom	Mean Square	F- value	Significance test
TPC-GA	Between Groups	10045	2	5022	30.38	0.00*
	Within Groups	11407	69	165
	Total	21451	71
TFC -CE	Between Groups	216	2	108	2.54	0.08
	Within Groups	2928	69	42.43
	Total	3143	71
TFC-QE	Between Groups	10333	2	5167	30.78	0.00*
	Within Groups	11581	69	168
	Total	21915	71

*Significant at p < 0.05 by using ANOVA test.

Fig 5

Variation of phenolic contents with error bar graphs based on their administrative zones at a confidence interval of 95%.

Pearson correlation matrix of teff samples variables (72 samples × 3 variables)

The Pearson correlation matrices employing correlation coefficient (r) for the samples were utilized to correlate the influence of total phenol content on the total flavonoid content by catechin and quercetin equivalents and measure the strength of a linear association between three variables, as shown in—Fig 6. The correlations between the total phenolic content and total flavonoid collected from the three administrative sampling areas of the teff samples were studied using linear regression correlation tests. The results are given in Fig 6. There was a positive correlation between total phenolic and total flavonoid content as catechin and quercetin equivalents. The variables are positively correlated and significant at (p = 0.01), as seen in the Pearson correlation matrix in, Fig 6, indicating that chemometric data treatment is possible. Among the various ways of multivariate analysis, hierarchical clustering can be applied to see the natural groupings of the samples and biplots to observe the relative positions of the variables and the distribution of the observations or data sets in a two-dimensional plot applied.

Fig 6

Pearson correlation matrix of teff samples variables (72 samples × 3 variables).

The 72 teff samples were trying to classify based on their geographical origin using hierarchical cluster analysis (HCA). In this classification model, the dendrogram was employed as a graphical tool to show the natural groupings or clusters based on the similarity ratios and Euclidean distances. As shown in Fig 7, the first and third clusters, encompassed by red and green rectangles, contain most of the samples obtained in the East Gojjam zone. Most teff samples from the West Gojjam zone were categorized on the last cluster or indicated by a violet color rectangle. The blue rectangle indicates the second cluster, containing teff samples from the Awi zone.

Fig 7

Dendrogram from agglomerative hierarchical cluster analysis of 72 teff samples using phenolic parameters.

On the other hand, samples obtained from the Awi zone did not show a clear cluster. The lack of clear grouping of the teff samples of this zone might be because teff samples from this zone are of different varieties. In addition, there might be a difference in agroecological parameters with the sampling sub-districts.

Based on the biplots in Fig 8, the two components explain up to 87.2% of the variation in the three administrative zones. Thus, from Fig 8, principal component 1 explains 67.2% of the total variability of polyphenol content of the teff grains. Principal component 2 accounts for 20% of the variability of the teff grains’ polyphenol content. The clusters with higher values of the principal component 1 are samples collected from West Gojjam and Awi zones, and principal component 2 is also associated with samples collected from the East Gojjam zone. Fig 8 shows a poor correlation between TFC-C.E and TFC-Q.E, while TPC demonstrates a higher correlation with TFC-Q.E. The distribution of the teff samples better cluster in a biplot based on their geographic origins. Most East Gojjam teff samples appeared between -2 and 1 in dimension1 (PC1) and slightly between -1 and 2 in dimension 2 (PC2). Fig 8 also demonstrates apparent clustering of the West and East Gojjam teff sample while samples collected from the Awi zone were intermixed with East Gojjam and West Gojjam zone. Most East Gojjam teff samples distribute at the left part, while those from West Gojjam are positioned at the right bottom side of the plot.

Fig 8

Biplot of the phenolic observations and variables based on geographic origins.

Conclusion

The total phenolic and flavonoid content in 72 teff samples collected from three administrative zones were successfully investigated using UV-Vis spectroscopy. The teff samples (Kuncho variety or white in color) collected from the West Gojjam zone were rich in TPC-G.A.E and TFC-Q.E content. In contrast, the same variety of teff samples from the East Gojjam zone exhibited lower total phenolic and flavonoid content. The phenolic content in teff samples from the Awi zone was arranged in the order of Brown (mixed) teff > Red teff > Tseday teff (white variety). These differences might be attributed to variations in the genetic make-up of the samples, soil chemistry, and agro-ecology of the sampling zones. The result from the analysis of variance revealed that the flavonoid content determined by catechin equivalent (TFC-C.E) exhibits no significant difference (p > 0.05) between the samples obtained from the three administrative zones. However, total polyphenol and flavonoid contents by quercetin equivalent (TPC-Q.E) showed a significant difference at p < 0.05 among samples of the West Gojjam and East Gojjam zones. Thus, based on this study, teff samples collected from the West Gojjam zone had a high total polyphenol content and quercetin equivalent flavonoids.

3 May 2022

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Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: The paper “Polyphenol and flavonoid content in major Teff [Eragrostis tef (Zuccagni) Trotter] varieties in Amhara Region, Ethiopia” provided interesting information about the polyphenol content of different Teff samples and varieties in relation to their region of provenience. For this aim a suitable number of samples was collected and analyzed.

I found the paper very interesting, however I have also identified some limitations that will be listed below:

- In the abstract the following sentence was reported “Such a difference is also evident in consumers' preference for teff grains from one location”. Report and explain better this consideration in “Results and discussions” or “Conclusions” paragraphs.

- In the introduction could be interesting to better explain the nutritional characteristics of Teef compared to other cereals: “Teff is gluten-free, abundant in minerals, vital amino acids, protein, and carbohydrates than other grains.”

- Explain the meaning of TTeff the first time that appeared

- Three different varieties of Teff were cited (and also analyzed). If the literature are reported differences, in term of phenolic compounds, between the Teff varieties (white, brown, and red) you could report them in the Introduction

- At the end of the introduction are cited, for the study, only two white Teff varieties

“Thus, this study was intended to quantify the level of total phenolic and total flavonoid contents in Kuncho teff (DZ-CR-387) and Tseday teff (DZ-CR-37) varieties collected from 21 sub-districts of three zones (East Gojjam, West Gojjam, and Awi)”

However, in Sample Collection and Pre-treatment paragraph were reported four different varieties:

“Four varieties of TTeff, such as Kuncho (white teff), Tseday (white teff), Brown (mixed), and Red teff were collected from model farmers in January 2020.”

Correct this difference and better explain the meaning of mixed for Brown Teff.

- In this study the determination of total flavonoid content was conducted expressing the results as catechin or quercetin equivalents.

In materials and methods was specify that some flavonoids such as quercetin, rutin, and galangin prefer to form a complex with aluminium chloride in an acid medium, therefore this class of flavonoids, was estimated in acidic conditions. However, in the description of method the used of acid medium was not reported. Pękal and Pyrzynska (2014) reported that acid or acetate solution could be used but, in some cases, only methanol or water was added. Correct or better explain the incongruity between the theory and the method applied.

Pękal A, Pyrzynska K. Evaluation of aluminium complexation reaction for flavonoid content assay. Food Anal Methods. 2014;7(9):1776-82.

- Some contradictions were observed between the text (in the results paragraph) and Tables 1 and 2; for example:

“On the other hand, the phenolic content recorded in samples from the West Gojjam and East Gojjam zones were ranged between 46.2 -92.2 mg/100 g and 45.95-128.5 mg/100 g, respectively.” The two zones were inverted and the data “45.95” was not reported in the Table 1.

“Comparing the mean value of TPC in samples from the three administrative zones, the highest average value of polyphenols was noted in the teff samples from the West Gojjam zone (95.04 mg G.A.E./100 g), followed by the Awi zone (79.37 mg G.A.E./100 g), and East Gojjam zone (71.39 mg G.A.E./100 g) (Table 2).” The data 79.37 and 71.39 don’t correspond to values reported in table 2

Carefully check all results.

- The sentence: “The lowest and highest mean concentrations of TFC were found in teff samples from East Gojjam (15.45 mg Q.E./100g) and the Awi zone (113.12 mg Q.E./100g), respectively, based on the mean value of total flavonoids at each sampling zone.” The results reported are not the mean concentrations listed in Table 2.

- Explain better how authors have conclude that:

“This implies that brown or mixed teff variety (from Awi zone) contained a higher total phenolic content than Kuncho teff variety (from East Gojjam zone) and Tseday teff variety (from West Gojjam zone). “(paragraph of Results and Discussion)

“the phenolic content in different teff samples was obtained in Brown teff > Kuncho teff > Tseday teff > Red Teff.” (paragraph of conclusions)

Overall, the manuscript is relatively well written however there are some typing and punctuation errors. For example, in Table 2, the Standard Error of the mean (indicated as SEM in the text), was reported as SE in the table and ESE in the legend.

Reviewer #2: Ref. No.: PONE-D-22-08028

Title: Polyphenol and flavonoid content in major Teff [Eragrostis tef (Zuccagni) Trotter]

varieties in Amhara Region, Ethiopia

The manuscript deals with a topic that could be of interest limited to the comparison of the polyphenol and flavonoid content in major Teff [Eragrostis tef (Zuccagni) Trotter] varieties in Amhara Region (Ethiopia).

The manuscript in some parts lacks clarity and an accurate revision is mandatory.

I have some questions and comments listed below:

Abstract:

the abstract is well organized.

In the line 3: ‘ ‘ region’s, please remove symbols.

Makeup: make-up

Last sentence of the abstract could be improved: “ These two dimensions (PC1 and PC2) explained the 87.2% of the total variance of the dataset”.

Introduction:

the introduction section is very rich but sometimes not clear and not presented in a logical way. Please revise this part.

“Vital” amino acids: please replace with essential.

“Its flour is mainly used to make "injera," Ethiopians' favourite national dish”: this has been said before, it is a repetition. Please modify in the manuscript.

“light to dark brown”: please modify “light brown to dark brown”.

“Reports indicate that total flavonoids are determined using two approaches: flavonols and flavones are selectively determined at 410–430 nm by reacting the sample solutions with AlCl3 in acidic or acetate solution. In contrast, flavan-3-ols (catechins and others), luteolin, and rutin could selectively react with AlCl3 solution in sodium nitrite and alkaline conditions, and measurement was done at 510 nm [24].

However, to the best of our knowledge, we did not find reports comparing the level of flavonoids determined by the two approaches simultaneously, either in teff or other products. Instead, the former method is most frequently reported to determine the total flavonoid content in various products”: this part should not be included in the introduction. Please move it to the discussion section.

“As information obtained from Amhara Region's Bureau of Agriculture, about 42 improved varieties of teff have been registered in the country, but only a few are widely cultivated in the region. Different types of teff are produced in all region zones”: In my opinion, this sentence is not so clear. Please better explain in the text.

Materials and Methods:

Please improve the resolution of Figure 1. In the map shown at the top left the names are not clearly legible. The sampling administration districts highlight in green in the maps are not visible.

In “Chemicals, Reagents, and Instruments”: ”Cary 60” Agilent, not Carry.

The Wetted sample: please remove the capital letter.

(porta centrifuge, Japan): please add the specifications of the centrifuge used in the previous paragraph.

and reported as mg gallic acid equivalent (mg G.A.E./100 g) of samples.

In “ Determination of total phenolic content by Folin–Ciocalteu assay”: please modify last sentence: “and reported as mg gallic acid equivalent for 100 g of samples (mg G.A.E./100 g).

Before reference [24], please remove the round bracket.

In “Determination of total flavonoid content as catechin equivalent“: “All measurements were performed in triplicate readings”. Were no analytical replicas of the spectrophotometric test made?

In “Determination of total flavonoid content as quercetin equivalent“: “All measurements were performed in triplicate readings”. Were no analytical replicas of the spectrophotometric test made?

Were quartz cuvettes used for the spectrophotometric readings? with which optical path? Please detail in the manuscript.

Statistical data analysis: Please express the acronyms TPC and TFC, TFC-QE first in the text.

Results and Discussion:

Figure 3: Concerning the calibration curve of catechin, the range of linearity was too short (abs from 0.00 to 0.2). Why were not considered standard solutions with higher concentration?

“The total phenolic content (TPC) is recorded as a milligram of gallic acid equivalent per 100 g of teff sample (mg of G.A.E./100 g teff). In addition, the catechin-like and quercetin-like total flavonoid contents are reported as milligram of catechin equivalent per 100 g of teff sample (mg of C.E./100 g) and milligram of quercetin equivalent per 100 g of teff sample (mg of Q.E./100 g teff) respectively”: this has already been said in materials and methods. Please remove it from Results and Discussion section.

Table1: please improve the formatting of table 1.

Figure 5 and Table 2 gives us the same information. Please remove Table 2 from the text (it could be added as supplementary materials). In Figure 5 please add error bars.

In the Discussion, Authors said that “The ANOVA test confirmed significant variations (P < 0.05) in its phenolic content among the 72 teff samples”. Where are reported the ANOVA results?

Table 3 is not clear. Please better explain in the text. Moreover, Principal component analysis has been applied but is not explain (in material and method section) and is not adequately discussed. This part needs to be improved.

Please check all the manuscript because there are several formatting errors. Please proofread the English, too.

**********

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Reviewer #1: No

Reviewer #2: No

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3 Jun 2022

We are very thankful to the reviewers and Editors for their appreciation of our manuscript entitled ' Polyphenol and flavonoid content in major Teff [Eragrostis tef (Zuccagni) Trotter]varieties in Amhara Region, Ethiopia, and the constructive criticism on certain issues. We are very grateful for your comments and feel that they added a lot of value and clarity to the overall organization of the manuscript. We agree with the reviewers and Editors that the comments raised should be addressed properly for better quality and acceptability of the manuscript. Therefore, we have revised our manuscript according to the reviewers' and editor’s comments, and the changes that have been made in the revised manuscript are track changed with different font colours.

The replies to the Reviewers' and editor’s comments are given carefully below:

Response to the Editor's comments

Comments #1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Response # 1: We have checked and confirmed our manuscript against the journal's guideline

Comment # 2. We note that the grant information you provided in the 'Funding Information' and 'Financial Disclosure' sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the 'Funding Information' section.

Thank you for stating the following in the Acknowledgments Section of your manuscript:

"The authors are thankful for the office for research and community service vice president of the Bahir Dar University for the financial support. In addition, Chaltu Reta is grateful to the college of the science of Bahir Dar University for sponsoring her PhD study."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"Authors received no special fund from the university except providing laboratory facilities and sampling costs."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Response # 2: We thank the Editor for raising this issue. We wish we could include the grant number in the funding statement. But we can't because we did not receive a formal fund from the university; rather, the research and community service vice president office of the university has covered costs related to sample collection, while the college of science provided the laboratory facilities because Chaltu Reta, a staff member of Bahir Dar University, is one of the students sponsored by the university. Thus, there was no grant number for this support and scholarship. Therefore, we have removed funds-related issues from this submission.

Comment #3: Please amend your list of authors on the manuscript to ensure that each author is linked to an affiliation. Authors' affiliations should reflect the institution where the work was done (if authors moved subsequently, you can also list the new affiliation stating "current affiliation:…." as necessary).

Response #3: Thank you so much for pointing out our mistake. We have now made the necessary corrections.

Comment # 4: We note that Figure 1 in your submission contain map/satellite images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

Response # 4: We have now prepared our map using the GPS points obtained from the sampling districts and sub-districts.

Comment #5: Please include a separate caption for each figure in your manuscript.

Response # 5: We have included a separate figure caption in the manuscript where the respective figure will be placed.

Comment # 6: Additional Editor Comments:

The paper needs careful revision. Authors are requested to respond to the individual comments of the reviewers. Please pay special attention to the statistical analysis

Response # 7: We have appreciated this comment. We have edited the manuscript considering the issues raised by the Editor and our respected reviewers. In addition, we have read the manuscript page by page and made substantial corrections.

Response to Reviewer: 1

Comment # 1: - In the abstract the following sentence was reported "Such a difference is also evident in consumers' preference for teff grains from one location". Report and explain better this consideration in "Results and discussions" or "Conclusions" paragraphs.

Response #1: We thank the Reviewer for his/her comment. As per the Reviewer's suggestion, we have removed such a sentence from the abstract section.

Comment # 2: In the introduction could be interesting to better explain the nutritional characteristics of Teff compared to other cereals: "Teff is gluten-free, abundant in minerals, vital amino acids, protein, and carbohydrates than other grains."

Response #2: In the introduction section, we have included the following paragraph that compares the nutritional composition of teff as compared to other common cereals.

Teff has a naturally higher nutritional value and is rich in essential amino acids and minerals such as Fe, Zn, Mg, and Ca compared to cereals such as maize and pearl millet. On the other hand, a comparable level of protein and carbohydrates is reported in teff grains compared to the most common cereal grains such as wheat, barley, sorghum, and pearl millet, and it is a gluten-free cereal grain [3, 5]. As many people are diagnosed with celiac disease and other forms of gluten sensitivity, the demand for gluten-free meals is increasing. As a result, the nutrient profile of teff grain suggests that it has a lot of potential for application in foods and beverages around the world [2, 6].

Comment # 3: Explain the meaning of TTeff the first time that appeared?

Response #3: Thank you for this comment. We have now defined the term "Teff" at the beginning (in the 1st lines of the abstract and introduction sections)

Comment # 4: Three different varieties of Teff were cited (and also analyzed). If the literature reported differences, in terms of phenolic compounds, between the Teff varieties (white, brown, and red) you could report them in the Introduction.

Response # 4: We have now included a paragraph showing the difference in nutritional composition between the three coloured varieties of teff

"Different teff varieties are on the market. In terms of color, the most common teff varieties are white, brown and red. Brow teff is also known as mixed colored teff. It contains natural mixed white and red coloured teff grains. Report indicated that there is significant variation in minerlas and nutritional constituents between the three colored variants [ 14, 15]."

Comment # 5: "Thus, this study was intended to quantify the level of total phenolic and total flavonoid contents in Kuncho teff (DZ-CR-387) and Tseday teff (DZ-CR-37) varieties collected from 21 sub-districts of three zones (East Gojjam, West Gojjam, and Awi). However, in Sample Collection and Pre-treatment paragraph were reported four different varieties: "Four varieties of TTeff, such as Kuncho (white teff), Tseday (white teff), Brown (mixed), and Red teff were collected from model farmers in January 2020." Correct this difference and better explain the meaning of mixed for Brown Teff.

Response # 5: The comments are accepted. We have now clarified the confusion. Such change can be depicted from the last page of the introduction section ( page 4)

Comment # 6: In this study the determination of total flavonoid content was conducted expressing the results as catechin or quercetin equivalents. In materials and methods was specify that some flavonoids such as quercetin, rutin, and galangin prefer to form a complex with aluminium chloride in an acid medium, therefore this class of flavonoids, was estimated in acidic conditions. However, in the description of method the used of acid medium was not reported. Pękal and Pyrzynska (2014) reported that acid or acetate solution could be used but, in some cases, only methanol or water was added. Correct or better explain the incongruity between the theory and the method applied. Pękal A, Pyrzynska K. Evaluation of aluminum complexation reaction for flavonoid content assay. Food Anal Methods. 2014;7(9):1776-82.

Response #6: Thank you so much for pointing out the fault we made. We have clarified the confusion and edited the section dealing with flavonoid analysis.

Comment # 7: Some contradictions were observed between the text (in the results paragraph) and Tables 1 and 2; for example: "On the other hand, the phenolic content recorded in samples from the West Gojjam and East Gojjam zones were ranged between 46.2 -92.2 mg/100 g and 45.95-128.5 mg/100 g, respectively." The two zones were inverted and the data "45.95" was not reported in Table 1.

Response#7: We appreciate our esteemed Reviewer's insightful view. The issues raised are fully noted, and we have corrected them in the reviewed manuscript.

Comment # 8. Explain better how authors have conclude that: "This implies that brown or mixed teff variety (from Awi zone) contained a higher total phenolic content than Kuncho teff variety (from East Gojjam zone) and Tseday teff variety (from West Gojjam zone). "(paragraph of Results and Discussion) "the phenolic content in different teff samples was obtained in Brown teff > Kuncho teff > Tseday teff > Red Teff." (paragraph of conclusions)

Response #8: comments were accepted and incorporated into the revised manuscript. We have clarified those confusions in the abstract section and the result and discussion section.

Comment # 9: Overall, the manuscript is relatively well written however there are some typing and punctuation errors. For example, in Table 2, the Standard Error of the mean (indicated as SEM in the text), was reported as SE in the table and ESE in the legend.

Response # 9: Thank you for your wonderful comments. We have read the manuscript page by page and significantly improved the typographic and spelling errors.

Reviewer 2.

Abstract:

Comment #1: In the line 3: '‘ region's, please remove symbols.

Response #1: We are very grateful to the Reviewer for pointing out this comment. The symbols have now been removed.

Comment #2: Makeup: make-up

Response #2: Thank you so much. We have converted the word “ Makeup” to “make-up”

Comment # 3: The Last sentence of the abstract could be improved: "These two dimensions (PC1 and PC2) explained the 87.2% of the total variance of the dataset".

Response #3 We thank the reviewers for pointing out this suggestion. We have now removed the stated sentence.

Introduction

Comment # 4: The introduction section is very rich but sometimes not clear and not presented in a logical way. Please revise this part

Response #4 We thank the Reviewer comment for his/her comment. We have thoroughly edited the introduction section and other sections of the manuscript.

Comment #5: "Vital" amino acids: please replace them with essential.

Response #5: We have replaced the phrase “ Vital amino acids” with “essential amino acids”.

Comment # 6: "Its flour is mainly used to make "injera," Ethiopians' favorite national dish": this has been said before, it is a repetition. Please modify the manuscript.

Response #6: We thank our Review for this comment. We have deleted this repeated sentence.

Comment #7: "light to dark brown": please modify "light brown to dark brown".

Response #7: We have accepted the comment and corrected it accordingly.

Comment # 8. Reports indicate that total flavonoids are determined using two approaches: flavonols and flavones are selectively determined at 410–430 nm by reacting the sample solutions with AlCl3 in acidic or acetate solution. In contrast, flavan-3-ols (catechins and others), luteolin, and rutin could selectively react with AlCl3 solution in sodium nitrite and alkaline conditions, and measurement was done at 510 nm [24].

However, to the best of our knowledge, we did not find reports comparing the level of flavonoids determined by the two approaches simultaneously, either in teff or other products. Instead, the former method is most frequently reported to determine the total flavonoid content in various products": this part should not be included in the introduction. Please move it to the discussion section.

Response #8: We thank our esteemed Reviewer for the suggestion. We took the following paragraph to the result and discussion section.”

“ In this study, the total flavonoids in the 72 teff samples were determined following two approaches. Reports indicate that not all flavonoids react with AlCl3 in the presence of sodium nitrite and sodium hydroxide. Instead, flavonols and flavones classes of flavonoids such as quercetin, quercetrin, galangin, and rutin, are selectively determined at 410–430 nm by reacting the sample solutions with AlCl3 in acidic, acetate buffer, or methanolic solutions. In contrast, flavan-3-ols (catechins and others) could selectively react with AlCl3 solution in sodium nitrite and alkaline conditions, and measurement can be done at 510 nm[27]. Thus, flavonoid data obtained with only one of the approaches could not reflect the total falvonoids content in a given sample”.

Comment # 9: "As information obtained from Amhara Region's Bureau of Agriculture, about 42 improved varieties of teff have been registered in the country, but only a few are widely cultivated in the region. Different types of teff are produced in all region zones": In my opinion, this sentence is not so clear. Please better explain in the text.

Response # 9: We appreciate the Reviewer’s comment and concerns. We have now clarified the stated paragraph as follows:

“As information obtained from Amhara Region's Bureau of Agriculture, about 42 improved varieties of teff have been registered in the country, but only a few are widely cultivated in the region.. Among the varieties of teff, Kuncho teff (DZ-CR-387) and Tseday teff (DZ-CR-37) are white by color and widely cultivated in the northwestern part of the Amhara region. Kuncho teff is cultivated in East Gojjam and West Gojjam zones, while Tseday teff variety is mainly grown in the Awi zone. In addition, red teff and brown teff varieties are also cultivated in the region, but consumers less prefer these colored varieties. Brown (mixed) teff is a variety of teff that is locally called “sergegna” and naturally exists asa mixture of white and red teff varieties. Kuncho and Tseday teff varieties have a high market price and are most preferred by consumers. But the same variety of teff produced in different zones and districts have different qualitiesand market prices, hence having consumer preferences. Since the Amhara region is one of the most teff-producing areas of the country, there is no report on the level of total phenolics and flavonoids in different teff varieties cultivated in this region”

Materials and Methods:

Comment #10: Please improve the resolution of Figure 1. In the map shown at the top left the names are not clearly legible. The sampling administration districts highlight in green in the maps are not visible.

Response #10: Thank you for the comment. Figure 1 has been replaced with a well-resolved figure.

Comment #11: In "Chemicals, Reagents, and Instruments":" Cary 60" Agilent, not Carry.

Response #11: It is corrected accordingly. Thank you once again.

Comment #12: The Wetted sample: please remove the capital letter.

Response # 12: It is corrected accordingly. Thank you for the comment

Comment #13: (porta centrifuge, Japan): please add the specifications of the centrifuge used in the previous paragraph.

Response # 13: A specification for the centrifuge is added in the materials and method section.

Comment #14: In "Determination of total phenolic content by Folin–Ciocalteu assay": please modify last sentence: "and reported as mg gallic acid equivalent for 100 g of samples (mg G.A.E./100 g).

Response #14: It is corrected accordingly.

Comment #15: Before reference [24], please remove the round bracket.

Response #15: Thank you, the round bracket before [24], is removed.

Comment #16: In "Determination of total flavonoid content as catechin equivalent ": "All measurements were performed in triplicate readings". Were no analytical replicas of the spectrophotometric test made?

Response #16: We thank our esteemed Reviewer. The sentence was confusing. We have now clarified it as follows in the revised manuscript.

“Each sample was analysed in triplicate.”

Comment #17: In "Determination of total flavonoid content as quercetin equivalent ": "All measurements were performed in triplicate readings". Were no analytical replicas of the spectrophotometric test made?

Response #17: thank you for your comment. We have corrected it as follows:

“Each sample analysed in triplicate”

Comment #18: Were quartz cuvettes used for the spectrophotometric readings? with which optical path? Please detail in the manuscript.

Response #18: Thank you for pointing out this comment. A description about the sample holder is given under the “ Chemicals, reagent and instrument section” as follows:

“. Quartz cuvette with 1 cm path length was as sample holder”.

Comment #19: Statistical data analysis: Please express the acronyms TPC and TFC, TFC-QE first in the text.

Response #19: Acronyms of TPC, TFC-Q.E and TFC-C.E have now been defined in the method section and statistical data analysis section.

Comment #20: Figure 3: Concerning the calibration curve of catechin, the range of linearity was too short (abs from 0.00 to 0.2). Why were not considered standard solutions with higher concentrations?

Response: We would like to appreciate our Reviewer’s comment. Before the calibration curve range was fixed, an initial screening test was performed for some samples. Then it was noted that the samples absorbance values were lower than 0.2. Then we decided to take several concertation of catechin standards that provide an absorbance value between 0.00 and 0.20. Otherwise, the linear range might go beyond this range.

Comment #21: "The total phenolic content (TPC) is recorded as a milligram of gallic acid equivalent per 100 g of teff sample (mg of G.A.E./100 g teff). In addition, the catechin-like and quercetin-like total flavonoid contents are reported as milligram of catechin equivalent per 100 g of teff sample (mg of C.E./100 g) and milligram of quercetin equivalent per 100 g of teff sample (mg of Q.E./100 g teff) respectively": this has already been said in materials and methods. Please remove it from the Results and Discussion section.

Response# 21 Thank you so much. The redundant sentence has been deleted from the revised manuscript.

Comment # 22. Table1: please improve the formatting of table 1.

Response# 22: We have appreciated the comment. We have now improved the formatting of table 1.

Comment # 23. Figure 5 and Table 2 gives us the same information. Please remove Table 2 from the text (it could be added as supplementary materials). In Figure 5 please add error bars.

Response# 23: Table 2 was removed, and the error bar was added to the graph in the revised manuscript.

Comment # 24: In the Discussion, Authors said that "The ANOVA test confirmed significant variations (P < 0.05) in its phenolic content among the 72 teff samples". Where are reported the ANOVA results?

Response# 24:Thank you for your comments, and the ANOVA results have now been incorporated into the revised manuscripts ( page 16).

Comment # 25. Table 3 is not clear. Please better explain in the text. Moreover, Principal component analysis has been applied but is not explained (in the material and method section) and is not adequately discussed. This part needs to be improved.

Response# 25: We are thankful to our respected Reviewer for raising this issue. We have significantly improved the discussion part of table 3, and the PCA. We have also explained the PCA in the method section.

Comment # 26. Please check all the manuscript because there are several formatting errors. Please proofread the English, too.

Response# 26: We have gone through the manuscript line by line and significantly revised the manuscript.

Submitted filename: Response to reviewers comments June 2022.docx

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30 Jun 2022

Submitted filename: Review_2.docx

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5 Jul 2022

Replies to the technical comments of the reviewers and Editors

Ref. No.: PONE-D-22-08028

Title: Polyphenol and flavonoid content in major Teff [Eragrostis tef (Zuccagni) Trotter]

varieties in Amhara Region, Ethiopia

We are very thankful to the reviewers and Editors for their appreciation of our manuscript entitled Polyphenol and flavonoid content in major Teff [Eragrostis tef (Zuccagni) Trotter]varieties in Amhara Region, Ethiopia, and the constructive criticism on certain issues. The replies to the Reviewers' and editor's comments are given carefully below:

Response to reviewer #1 comments.

Comments #1: Some little typing and punctuation errors were still observed (e.g. in the tile and in the test lacking spaces)

Response #1: Significant typographic errors and punctuation corrections have been taken throughout the manuscript.

Comments #2: In the Introduction replace "It's a chasmogamous C4 annual cereal with a fibrous…" with "Teff is a chasmogamous C4 annual…";

Response #2: The reviewer's suggestion is incorporated in the revised manuscript.

Comment #3: In the Results and Discussion, Figure 1 on gallic acid results is Figure 2;

Response # 3: Figure 1 refers to a map of the sampling areas, while Figure 2 refers to the gallic acid standard's calibration curve and UV spectra.

Comment #4: In the Results and Discussion, I suggest to modify the following sentence "This implies that brown or mixed teff variety (from Awi zone) contained a higher total phenolic content than Kuncho teff variety (from East Gojjam and West Gojjam zone) and Tseday teff variety (from Awi zone)." Some samples from the West Gojjam zone have a high TPC, indeed the mean value of TPC and TFC-Q.E were found to be the highest in West Gojjam teff samples compared to the samples from other zones

Response #4: The brown or mixed teff variety from Awi zone contained a higher total phenolic content than the white teff (Tseday variety ) collected from the Awi zone. The mean TPC values were compared between the two white teff varieties (Kuncho and Tseday) collected from the three administrative zones. It was noted that the Kuncho teff variety collected from the West Gojjam Zone contained a higher mean TPC value than the same Kuncho variety collected from the East Gojjam zone and the other variety (Tsedey) from the Awi zone.

Comment #5: Among varieties from the Awi zone, the brown samples showed the highest TPC. Similar comments could be also arranged for the TFC.

Response #5: Thank you once again for pointing out this. We have modified the phrase as follow: Similar to the TPC value, the brown (mixed) teff samples from the Awi zone had higher flavonoid contents (Table 1) than the white variety from this sampling zone. Comparing the mean value of TFC between the three sampling zones, samples from the west Gojjam zone had a mean TFC-Q.E value higher than samples from East Gojjam and Awi zones

Comment #6: In the Results and Discussion, in the sentence "On the other hand, relatively comparable TPC values with the present study were reported by [14] for white and brown teff varieties collected from the Tigray region of Ethiopia." could be interest to specify the values reported by the reference authors

Response #6: Thank you for your comments. We have now incorporated the data reported by [14} as follows. On the other hand, 263 mg GAE/100g and 448 mg GAE /100g, respectively, for white (Tseday type) and brown teff varieties collected from the Tigray region of Ethiopia were reported by [14].

Comment #7: Table 3 must be insert in the text as a Figure or as a Table?

Response #7: We are grateful for this comment. We have changed table 3 into Figure 6. Then the previous figure 6 is labeled as figure 7, and the last figure 7 is captioned figure 8 in the revised manuscript. This change has been corrected in the text too.

Submitted filename: Response to the reviewer comments.docx

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12 Jul 2022

Polyphenol and flavonoid content in major Teff [ Eragrostis tef (Zuccagni) Trotte r] varieties in Amhara Region, Ethiopia

PONE-D-22-08028R2

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Reviewers' comments:

18 Jul 2022

PONE-D-22-08028R2

Polyphenol and flavonoid content in major Teff [Eragrostis tef (Zuccagni) Trotter] varieties in Amhara Region, Ethiopia

Dear Dr. Atlabachew:

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