Assessment and Determination of Bittering Agents, Essential Oils, and Antioxidants of Gesho ( Rhamnus prinoides L. Herit) Collected from Amhara Region, Ethiopia

Rhamnus prinoides (Gesho) is an angiosperm dioecious plant, which is used for cash income, as a bittering agent for the preparation of the local alcoholic beverage and medicinal values. Although it has intense use, still its practical application is limited for local alcoholic beverage preparation, and there is no suﬃcient scientiﬁc report on the level and quantity of bittering agents, essential oils, and antioxidants of this plant. Therefore, the objective of this research was to assess and determine the bittering agents, essential oils, and antioxidants of R. prinoides . The leaves of R. prinoides were collected from eleven diﬀerent sample sites and allowed to sun dry and then powdered. It was further dried in an oven at 60 ° C for 1h, and then, total resin, essential oils, alpha acid, iso-alpha acid, beta acid, ﬂavonoids, and polyphenols of R. prinoides were determined. All the data were analyzed using SPSS version 20.0. Based on this study ﬁnding, (16.03 ± 0.03–17.05 ± 0.04%) total resins, (10.00 ± 0.08–11.23 ± 0.07%) soft resins, (5.65 ± 0.11–6.40 ± 0.35%) hard resins, (1.82 ± 0.11–3.14 ± 0.02mg/l) alpha acids, (2.26 ± 0.15–4.15 ± 0.10mg/l) iso-alpha acids, (2.62 ± 0.04–4.29 ± 0.08mg/l) beta acids, (7.74 ± 0.11–12.47 ± 0.13%) essential oils, (23.00 ± 0.14–25.01 ± 0.09%) ﬂavonoids, and (2.11 ± 0.01–2.41 ± 0.02%) polyphenols were obtained in the leaves of R. prinoides. R. prinoides leaves taken from Tikledingay has shown statistically ( P ≤ 0 . 05) signiﬁcant amount of soft resin (11.23 ± 0.07%), alpha acid (3.14 ± 0.02mg/l), iso-alpha acid (4.15 ± 0.10mg/l), essential oils (12.47 ± 0.13%), ﬂavonoids (25.01 ± 0.09%), and polyphenols (2.41 ± 0.02%) in comparison with other samples. Generally, R. prinoides is a good source of bittering agents, essential oils, and antioxidants. However, the quantity of bittering agents, essential oils, and antioxidants in the leaves of R. prinoides was varied with sample collection sites. So R. prinoides can be used as a substitute for commercial hops used in the breweries, and promotion should be done to introduce the potential of Gesho as a bittering agent, source of aroma and ﬂavor, and also antioxidant for beer and other alcoholic beverages.


Introduction
Rhamnus prinoides, Gesho in Amharic, is a dicotyledonous angiosperm, which belongs to the genus Rhamnus under the family Rhamnaceae. It is commonly known as dark blinkblaar, dogwood, and shiny leaf [1]. R. prinoides is a dense shrub or small tree which grows up to 6 meters tall. It is one of the two species that represent the genus Rhamnus and a widespread plant species in Africa [2]. e plant is native to Ethiopia, Eritrea, Botswana, Namibia, Lesotho, the treatment of atopic dermatitis [6]. e fruits of this plant are used for ringworm infection treatment [3]. e different chemical compounds, such as musizin, sorigenin, geshoidin, chrysophanol, physcion, emodin, 10oxoprinoidin, quercetin, 3-O-methylquercetin, rhamnocitrin, and rhamnazin, were isolated from the leaves of R. prinoides. Of the various chemical substances found in R. prinoides, naphthalene glucoside named geshoidin is responsible for the bitter attributes of the plant [3]. Geshoidin is a nontoxic substance found in abundant quantities in the stem and leaves of R. prinoides [7]. R. prinoides is quite different from hops (Humulus lupulus) and is grown largely in Ethiopia and is currently available in the local market in the dried form [8]. For hundreds of years, it has been used as a bittering agent in the traditional brewing process in East Africa [9].
In Ethiopia, R. prinoides is mainly used to provide bitterness, a special aroma, and flavor for traditional alcoholic drinks [10,11]. e leaves and stems of the plant are used to impart the characteristic bitter taste to domestically produced beverages, such as Tella and Tej, and it has been estimated that more than 5 million people consume it every day [12]. Gesho leaves and stems are essential ingredients in the preparation of these traditional fermented beverages. e plant has been reported to regulate the microflora responsible for the fermentation process. It plays an important role in suppressing certain bacteria during the fermentation process [13].
Hop is the basic ingredient used for bittering, flavoring, and aroma imparting agent for beer production. Also, it has a bacteriostatic activity to inhibit the growth of most microorganisms [14]. R. prinoides can serve as a substitute for hops used in beer production [8].
Although R. prinoides has intense use, its practical application has been limited for traditional/local alcoholic beverages preparation [15]. Also, no sufficient previous scientific report was found on the level and quantity of bittering agents, essential oils, and antioxidants in the leaves of this plant cultivated in different parts of Ethiopia and elsewhere. Hence, to use the leaves of this plant as a bittering agent, sources of aroma and flavor, and also as an antioxidant in beer brewing and other commercial alcoholic beverages preparation, the assessment and determination of its different physicochemical characteristics are significant. erefore, the objective of this research is to assess and determine the bittering agents, essential oils, and antioxidants of Rhamnus prinoides (Gesho), collected from different cultivation places of the Amhara region, Ethiopia.

Description of the Study Area and Study Design.
e study area was included central Gondar Zone (Delgie, Gondar, Kola Diba, and Tikledingay), North Gondar Zone (Debark), West Gojjam Zone (Finote Selam), South Gondar Zone (Ambessamie, Debre Tabor, and Wereta), North Wollo Zone (Geregera), and Awi Zone (Injibara). e collected leaf samples were investigated in the Microbial and Organic Chemistry laboratory, University of Gondar, Ethiopia. It is located at a distance of 727 km away from Addis Ababa, the capital city of Ethiopia. In this study, an experimental research design was employed.

Sample Collection and Transportation.
A sampling of R. prinoides leaves was done using purposive sampling techniques. Fresh leaves of R. prinoides were collected and then sun-dried in the sample collection areas. e leaf samples collected were labeled and placed in polyethylene plastic bags. en, it was brought to the University of Gondar, Microbial, and Organic Chemistry laboratory for further pretreatment and analysis.

Sample Preparation.
e collected sun-dried R. prinoides leaves samples were cleaned manually to remove seeds, stalks, stones, and other extraneous materials. e sun-dried and cleaned leaves samples were crushed and pounded into small pieces of particle size of 0.75 mm and below with laboratory Miller. It was then further dried in an oven at 60°C for 1 h to remove the moisture content of leaves powder. e powdered leaf samples were stored in plastic bags in a cool and dry place, and then, laboratory analysis of the leaves sample was carried out to determine the bittering agents, essential oils, and antioxidants of R. prinoides.  (20) grams of the leaves sample of R. prinoides was dissolved in 100 ml of cold methanol (99.5%, Sisco Research Laboratories Pvt. Ltd, India) in a conical bottom flask, and the mixture was vigorously agitated by swirling the flask. ereafter, the solution was filtered using Whatman filter paper (No. 14). e filtrate containing the resin was then dried to constant weight over a water bath. e total resin was then calculated as a percentage of the original weight of the sample dissolved in methanol [16]. (20) grams of the R. prinoides leaves sample was dissolved in 20 ml of n-hexane (99.9%, Pentokey Organy Ltd, India) thoroughly stirred and filtered using Whatman filter paper (No. 14). e filtrate was dried to constant weight over a water bath. e soft resin was then calculated as a percentage of the original weight of the sample dissolved in n-hexane. A hard resin was determined by subtracting soft resin from total resin [17].

Iso-α-Acid Determination.
A 0.15% (w/v) solution of the leaves sample of R. prinoides was made using distilled water. e solution was boiled for 90 min and cooled and filtered using Whatman filter paper (No. 14). A 15 ml sample extract was acidified with 0.5 ml 6 N HCl (35-38%, Blulux laboratories Pvt. Ltd, India) and mixed with 15 ml of pure isooctane (99.5% Loba Chemie Pvt. Ltd, India) in a flask shaker, and 10 ml of the isooctane extract was washed with 10 ml of a mixture of methanol and 4 N HCl (68 : 32, v/v).

Alpha-and
Beta-Acid Determination. 0.15 grams of the sample was added in 100 ml cold methanol in a flask shaker. e solution was then centrifuged at 2500 rpm for 20 min, and then, decanted supernatant was acidified with 0.002 N HCl; its absorbance at 355, 325, and 275 nm was determined using a spectrophotometer and the α-acid calculated using [18] method: α-acid (mg/l) � 73.79 (A325) − 51.56 (A355) − 19.07 (A275) and Β-acid (mg/l) � 55.57 (A355) − 47.59 (A325) + 5.10 (A275), where A is the absorbance reading at the specified wavelength. (20) grams of the leaf sample of R. prinoides was placed inside a thimble and inserted into the inner tube of the Soxhlet extractor (BS2071, England).

Determination of Essential Oils of R. prinoides. Twenty
is apparatus was then fitted to a round-bottom flask, which contained 200 ml of n-hexane. A reflux condenser was also mounted and fitted on the apparatus. e set-up was held tight to a retort-stand and then placed on a suitable electric heating mantle (Type ER, E.E.C) that was switched on for an extraction time of 120 min at the boiling point of the solvent (n-hexane, 60°C). e vapor was passed up through the tube and condensed by the condenser, and the condensed solvent falls into the thimble and slowly fills the body of the Soxhlet. When the solvent reached the top of the tube, it siphoned over into the flask and thus removed the portion of the sample that has been extracted in the thimble. e solvent recovery process has been done using a rotary evaporator (RE20729, Bibby Sterilin Ltd, England). e mixture of solvent (n-hexane) and oil was heated in the flask over a heater. On constant heating, the solvent evaporated and thereafter condensed in the flask. e condensed solvent was collected into the flask. en, the extracted oil was recovered, and its mass was recorded and calculated as a percentage from the original sample [16].

Determination of Antioxidants of R. prinoides
2.6.1. Determination of Flavonoids. Ten (10) grams of the leaves sample of R. prinoides was extracted repeatedly with 100 ml of 80% aqueous methanol at room temperature. e whole solution was filtered through Whatman filter paper (No. 14). e filtrate was later transferred into a crucible and evaporated to dryness over a water bath and weighed to a constant weight using a sensitive electronic balance [19].

Determination of Polyphenols.
e sample was boiled with 50 ml of ether (99%, Blulux laboratories Pvt. Ltd, India) for the extraction of phenolic component for 15 min, 5 ml of the extract was pipetted into a 50 ml flask, and then 10 ml of distilled water was added. A 2 ml of ammonium hydroxide solution (Blulux laboratories Pvt. Ltd, India) and 5 ml of concentrated amyl alcohol (Blulux laboratories Pvt. Ltd, India) was also added. e sample was made up to the mark and left for 30 minutes for color development.

Statistical Analysis.
All the data were analyzed using the Statistical Package for Social Sciences (SPSS) version 20.0 program. Mean and standard deviation of the triplicate data were calculated using a one-way analysis of variance (ANOVA). Post hoc multiple comparisons, particularly the Duncan test, was employed to compare the significance between groups. e statistically significant differences were considered at P ≤ 0.05.
us, the amount of total resin content obtained from R. prinoides leaves collected from these sample sites were ranged from 16.03 ± 0.03 to 17.05 ± 0.04%) and found to be comparable with the total resin values of R. prinoides leaves (18.46 ± 0.03%) reported in [8]. And also, the authors in [21] reported that the amount of hop resin ranged from 12 to 24% with a mean value of 18.5%. In this case, the total resin content of the leaves of R. prinoides (16.03 ± 0.03-17.05 ± 0.04%) was found to be within the range of total resin values of air-dried commercial hop cones. e values of total resin (16.03 ± 0.03-17.05 ± 0.04%) obtained in this study from R. prinoides leaves collected from different sample sites were found less than the total resin values (20.4%) of Vernonia amygdalina reported in [16]. e total resins represent the sum of all bitter resins and can be further divided into hard and soft resins [22]. Hard resins are insoluble in hexane, and soft resins are soluble in hexane [23]. In this study, the amount of soft resins content obtained from leaves of R. prinoides collected from Debark, Gondar, Debre Tabor, Finote Selam, Wereta, Injibara, Kola Diba, Geregera, Delgie, Ambessamie, and Tikledingay were 10.00 ± 0.08%, 10.23 ± 0.27%, 10.30 ± 0.22%, 10.35 ± 0.15%, 10.36 ± 0.18%, 10.45 ± 0.08%, 10.46 ± 0.13%, 10.50 ± 0.16%, 10.52 ± 0.33%, 10.86 ± 0.10%, and 11.23 ± 0.07%, respectively (Table 1). ese values of soft resin ranged from 10.00 ± 0.08 to 11.23 ± 0.07% depending on sample collection sites and Journal of Chemistry were found to be lower than the soft resin values (15.73 ± 0.03%) of R. prinoides leaves reported in [8]. But, in this case, the result obtained for soft resin from R. prinoides leaves was found to be within the range of soft resin values of (10-18%) commercial hops, used for beer production reported in [21].
So the content of the essential oil obtained in R. prinoides leaves was in the range of 7.74 ± 0.11-12.47 ± 0.13% depending on the sample sites and higher than the content of the essential oil of dried commercial hop cones reported by several authors [21,24,26]. Berhanu [8] reported that the mean values of essential oils content of R. prinoides leaves Table 1: Average concentrations of the bittering components in R. prinoides leaves samples collected from different sample sites.

Sample sites Values Total resin (%)
Soft resin (%) were 1.13 ± 0.02%. So, the content of essential oils obtained in this case (7.74 ± 0.11-12.47 ± 0.13%) was greater than that of R. prinoides leaves reported in [8]. Here, one possible reason for such a difference may be due to the method used. e method used in this study for essential oil extraction and determination was completely different from the methods used in [8]. In this study, the quantity of essential oil from leaves (7.74 ± 0.11-12.47 ± 0.13%) powder of R. prinoides was higher than the content of the essential oil (1.20%) of V. amygdalina reported in [16]. A significant amount of essential oils was obtained in this study, and this indicates that the leaves of R. prinoides can be serving as a good source of aroma and flavor in the production of alcoholic beverages.
As it is shown in Table 3, the mean values of polyphenols obtained in this study from R. prinoides leaves collected from Debark, Finote Selam, Kola Diba, Delgie, Gondar, Debre Tabor, Geregera, Injibara, Wereta, Ambessamie, and Tikledingay were 2.11 ± 0.01%, 2.12 ± 0.015%, 2.15 ± 0.023%, 2.17 ± 0.061%, 2.18 ± 0.01%, 2.18 ± 0.06%, 2.27 ± 0.01%, 2.27 ± 0.07%, 2.32 ± 0.07%, 2.33 ± 0.06%, and 2.41 ± 0.02%, respectively. ese values of polyphenols ranged from 2.11 ± 0.01 to 2.41 ± 0.02%, depending on the sample collection sites, and were found within the range of the polyphenol values of commercial hop (2-5%), which was reported in [24]. And also, according to [21], the content of hop polyphenols ranged from 2 to 5% with a mean value of 3.5%. In this case, the content of polyphenols of leaves of R. prinoides (2.11 ± 0.0-2.41 ± 0.02%) was found to be within the range of commercial hops. e result obtained in this study suggests that R. prinoides leaves are a good source of polyphenols, and it may serve as an antioxidant for commercial alcoholic beverages brewing. e result obtained from this study showed that R. prinoides leaves are a good source of total resin, soft resin, hard resin, α-acid, β-acid, flavonoids, and polyphenol and were found to be comparable with that of commercial hops [21,24]. us, the amount and level of total resin, soft resin, hard resin, α-acid, β-acid, iso-α-acid, essential oils, flavonoids, and polyphenols of R. prinoides leaves collected from different sample locations were different. Such a difference and irregularities cannot be certainly explained; nevertheless, it might be attributed to a difference in geographical areas of plant sample collection, climate and weather conditions, soil types, and also a variety difference of the plant.

Conclusion
In this investigation, different physicochemical characteristics of R. prinoides leaves that were collected from different farmlands of the Amhara region, Ethiopia, were assessed and characterized. e results obtained from this study showed that the leaves of R. prinoides are good sources of bittering agents, essential oils, and antioxidants and were found in good agreement with most of the reported values of commercial hop found in the literature. However, the amount and level of bittering agents, essential oils, and antioxidants of R. prinoides leaves were varied with sample source sites. e factors that contributed to such variation are likely to be a difference in the plant geographical location, climatic and weather conditions, soil types, and variety difference. us, the result obtained in the present study can be used as a baseline to formulate bittering substance required for beer production and other alcoholic beverages.

Recommendation
Based on the results of this study, the following recommendations have been provided: (i) Gesho leaves are a good source of bittering agents, essential oils, and antioxidants and can be a substitute for commercial hops. (ii) Promotion should be done to introduce the potential of Gesho as bittering agents, source of aroma, and flavor for beer and other alcoholic beverages. (iii) Promoting further detailed investigations needed on the impact of soil type and growing environment on the quantity of bittering components, essential oils, and antioxidants of R. prinoides leaves.

List of Abbreviations
AOAC: Association of Official Analytical Chemists iso: Isomerize l: Liter mg: Milligram ml: Milliliter rpm: Revolution per minute.

Data Availability
Upon request, the data used to support the result of this study are available from the corresponding author.

Conflicts of Interest
e authors declare that they have no conflicts of interest.