LC-MS Analysis, Total Polyphenol Content and Antioxidant Activity of Ethanolic Fractions of Skin (Epicarp) and Seed Pulp (Mesocarp) of Green and Red Wild Africa Berries.

doi:10.21203/rs.3.rs-3226316/v1

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LC-MS Analysis, Total Polyphenol Content and Antioxidant Activity of Ethanolic Fractions of Skin (Epicarp) and Seed Pulp (Mesocarp) of Green and Red Wild Africa Berries.

https://doi.org/10.21203/rs.3.rs-3226316/v1

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Background

These green and red wild African berries (Lannea microcarpa) are not cultivated like other known crops but grow in the wild, mostly in the northern part of Nigeria. Although its culinary uses are quite popular during the early part of the rainy season, its chemistry remains vague, hence the present study.

Aim

The study aims at assessing the antioxidant activity of these berries and further identifying the phytocompounds to determine their biofunctional abilities.

Methods and Materials

The fresh fruits of green and red wild berries were collected and identified. The antioxidant activity of the berry types was assessed employing three assays (2-diphenyl-1-picrylhydrazil (DPPH) free radical scavenging assay, 2, 2 - azino-bis-(3-ethyl) benzothiazoline-6-sulfonic acid (ABTS) radical cation scavenging activity, hydrogen peroxide (H₂O₂) reducing power) of the skin and pulp of green and red wild African berries (FUS = Green Berries Skin, FRS = Red Berries Skin, FSP = Red Berries Seed Pulp, UFSP = Green Berries Seed Pulp). The total polyphenol content (TPC), total flavonoid content (TFC), and total anthocyanins (TAC) were also investigated. These ethanolic fractions of the extracts were evaluated employing Liquid Chromatography-Mass Spectrometry (LC-MS).

Results

Total anthocyanin content is interestingly higher in FRS and FSP (deep red berries) than in FUS and UFSP (green berries). In the DPPH assay, the antioxidant activity of the various parts of the two berry types followed the following trend: UFSP ˃ FUS ˃ FRS ˃ FSP and a similar trend was noticed in the ABTS assay. Interestingly, the H₂O₂ assay followed the following trend: FUS ˃ UFSP ˃ FRS ˃ FSP. About 21 compounds belonging to different phenolic families, namely anthocyanidins, flavones, flavonols, flavan-3-ols, flavanones, hydroxycinnamic acids, hydroxybenzoic acids, and hydroxycoumarins, were identified. Cyanidin-3-O-glucoside is reportedly present in all the parts investigated.

Conclusion

This study provided insight into the phytochemical profiles and pharmacological importance of L. microcarpa beyond its basic nutritional values. This fruit can be useful as a functional food.

Lannea microcarpa

LC-MS

Functional Foods

Wild berries

Quercetin

The oxidation reactions are necessary for life to continue. Consequently, similar reactions are involved in food metabolism as well as other biological activities. These reactions can generate free radicals, which can have deleterious consequences (Hakkinen et al., 1999; Astley, 2003). They cause the cell to be damaged. Antioxidants, such as warriors, play a critical role in preventing these harms. Antioxidant systems in living beings are complex and contain a variety of antioxidants. Antioxidants include vitamins C and E, glutathione, some enzymes such as catalyzes and superoxide dismutase, and some peroxides. Oxidative stress occurs when antioxidant levels drop or antioxidant enzymes are inhibited (Shaiban et al., 2006; Arabshahi-Delouee & Urooj, 2007). Many diseases, such as coronary heart disease, atherosclerosis, cancer, and aging, can be caused by reactive oxygen species. These disorders are caused by the unregulated synthesis of reactive oxygen species (ROS) and an imbalanced antioxidant defense system. Natural antioxidants derived from plants, as well as synthetic antioxidants such as BHA and BHT, can be used to reduce the damaging effects of ROS (Li et al., 2008). However, there are some hazards associated with using these compounds. As a result, the use of synthetic antioxidants has been prohibited in many nations in the last few years, and demand for natural antioxidants has grown (Gülçin et al., 2006). As a result, the majority of people generally prefer antioxidants to synthetic antioxidants (Sun & Fukuhara, 1997).

Fruit intake has been linked to a variety of health benefits, the majority of which are attributed to phytochemical components found in fruits. Among these, phenolic compounds have received the greatest attention due to their antioxidant properties and promise for cancer, inflammatory, and cardiovascular disease prevention (Bello et al., 2020; Julie et al., 2005). In this context, there is a continuing quest for underutilized fruits, such as wild berries (both green and red varieties), which grow in the wild and are consumed mostly by local communities but have been recommended as fruits of increasing commercial importance. Polyphenols have been linked to various antioxidant activities.

There is a tree named Lannea microcarpa Engl & K. Krauss (Anacardiaceae), which is popularly found in the Sahelian-Sudan areas of Africa. This tree usually produces fruits that are termed "wild," "neglected, or "African berries." These wild berries are mostly deep wine or green in color and are mostly consumed by the locals; they are used in the production of local juices and employed in managing some diseases, i.e., cough, rickets, and scurvy (Bationo et al., 2012; Arbonnier, 2002). Anthocyanins and tannins have been confirmed present in the wild berries epicarp’s extract of L. microcarpa, further fractionation and investigation of the polar extract of the fruit epicarp allowed the detection of myricetin 3–O-α-_l- rhamnopyranoside, myricetin 3-O-β-d-glucopyranoside, 4'-methoxy-myricetin 3-O-α-_l-rhamnopyranoside, isovitexin, vitexin, epi-catechin, gallic acid (Picerno et al., 2006; Pale, 1998). A study to ascertain and compare the polyphenolic contents, antioxidant activity and identify the phytocompounds of the wild/African berries skin (epicarp) and seed pulp (mesocarp) is the thrust of this work. The work intends to identify new and interesting foods and supplements from wild fruits, i.e., berries, and other food plants found in the West Africa region.

2.1. Collection

The red and green African berries were collected from the surroundings of Katsina Metropolis, Katsina State, Nigeria in May 2021 which is the largest local government in the state. It is situated at Latitude N120 59’27.47” and Longitude E70’36’14.81”. Katsina is located 260 km east of the city of Sokoto and 135 km Northwest of Kano, close to the border with Niger Fig. 2. The city is the center of an agricultural region and the population of the city is mainly from the Fulani and Hausa ethnic groups. The samples were identified in the Department of Biological Science, Federal University, Dutsin-Ma, Katsina State. The samples were separated, washed, and air-dried under laboratory conditions. Afterward, the dried samples were then pulverized by the use of a wooden mortar and pestle and thereafter stored in a polythene bag pending further use.

2.2 Extraction

These pulverized samples (berries skin, and seed pulp for the two berries types) were exhaustively extracted with 100% ethanol using the percolation extraction method. Almost 120 g of the different pulverized samples were weighed in duplicate into different extraction bottles and 400 mL of ethanol was added to each of them until they were soaked. The extracts had different initial colours. The extraction was carried out using the percolation extraction method and then filtered into new different sample bottles. The extracts were concentrated in vacuo using a rotary evaporator below 100°C. The acquired extracts were thereafter air-dried at room temperature in an evaporating basin.

2.3 Quantification

2.4 Total polyphenolic content

The Folin-Ciocalteau method as suggested by Singleton et al. (1999) was employed to spectrophotometrically determine the total contents of polyphenolic present in the polyphenolic-rich extracts (Termote et al., 2001; Dicko et al., 2002; Bello et al., 2020). Data used for analysis were collected in triplicate. The standard control used in this determination is Gallic acid and results were expressed as mg gallic acid equivalent per gram sample. The total polyphenol was reported.

2.5 Total flavonoid content

The aluminum chloride colorimetric method was used for the determination of the total flavonoid content of the sample. For total flavonoid determination, quercetin was used to make the standard calibration curve. Stock quercetin solution was prepared by dissolving 5.0 mg quercetin in 1.0 mL methanol, then the standard solutions of quercetin were prepared by serial dilutions using methanol (5–200 µg/mL). An amount of 0.6 mL diluted standard quercetin solutions or extracts was separately mixed with 0.6 mL of 2% aluminum chloride. After mixing, the solution was incubated for 60 min at room temperature. The absorbance of the reaction mixtures was measured against blank at 420 nm wavelength with a Varian UV-Vis spectrophotometer (Cary 50 Bio UV-Vis Spectrophotometer, Varian). The concentration of total flavonoid content in the test samples was calculated from the calibration plot (, ) and expressed as mg quercetin equivalent (QE)/g of dried plant material. All the determinations were carried out in triplicate.

2.6 Total Anthocyanins

The total anthocyanins content in the extract was estimated using the colorimetric method based on a Trim-Hill reaction. The extract (0.1 g) was mixed with 4 mL of Trim-Hill reagent. Trim-Hill Reagent comprises acetic acid, copper II sulfate, and concentrated hydrochloric acid 10:1:0.5. Acetic acid (5%), CuSO₄ (10%), HCl (5%) (Trim-Hill, 1952). Then, the mixture was developed at room temperature with occasional shaking for 15 min. The absorbance was measured at 609 nm and the blue color indicated the presence of anthocyanins. The amount of anthocyanins was calculated using an aucubin (0.1–1 mg/mL) calibration curve (Termote et al., 2001).

2.7. Biological Activity

2.8. Antioxidant activity

2.9. 2-diphenyl-1-picrylhydrazil (DPPH) free radical scavenging assay

DPPH is one of the main assays used in assessing the antioxidant activity of plant material employed in this study. The assay was carried out in harmony with the method described by Bello et al., 2018a; Bello et al., 2018b; Bello et al., 2018c;

2.10. 2,2-azino-bis-(3-ethyl)benzothiazoline-6-sulfonic acid (ABTS) radical cation scavenging activity

This method was carried out employing the method of Bello et al. (2018a). Data used for analysis were collected in duplicate (Bello et al., 2018a).

2.11. Statistical analysis

All the analyses concerning the total antioxidant activity were carried out in the duplicate analysis. However, the other analyses were carried out in triplicate. The data were recorded as mean ± standard deviation and analyzed by SPSS

2.12. Liquid Chromatography-Mass Spectrometry (LCMS)

2.13. Protocol for LCMS Analysis (Generic Method) using LC Waters e2695 separation module with W2998 PDA and couple to ACQ-QDA MS

The samples were analyzed using liquid chromatography (LC) tandem mass spectrophotometer (MS) as described by (Piovesana et al., 2018) with some modifications. The extracted samples were reconstituted in methanol and filtered through a polytetrafluoroethylene (PTFE) membrane filter with 0.45 µm size. After filtration, the filtrate (10.0 µl) was injected into the LC system and allowed to separate on Sunfire C18 5.0 µm 4.6 mm x 150 mm column. The run was carried out at a flow rate of 1.0 mL/min, with Sample and Column temperature at 25^oC. The mobile phase consists of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) with a gradient as below

Time	% A	% B
0	95	5
1	95	5
13	5	95
15	5	95
17	95	5
19	95	5
20	95	5

The ratio of A/B 95:5 this ratio was maintained for a further 1 min, then A/B 5:95 for 13 min, to 15 min. then A/B 95:5 to 17 min, 19 min, and finally 20 min. the PDA detector was set at 210–400 nm with a resolution of 1.2 nm and a sampling rate of 10 points/sec. The mass spectra were acquired with a scan range from m/z 100–1250 after ensuring the following settings: ESI source in positive and negative ion modes; capillary voltage 0.8 kv (positive) and 0.8 kv (negative); probe temperature 600^oC; flow rate 10 mL/min; nebulizer gas, 45 psi. MS is set in automatic mode applying a fragmentation voltage of 125 V (Piovesana et al., 2018). The data was processed with Empower 3. The compounds were identified based on the following information, elution order, retention time (Rt), fragmentation pattern, and Base m/z.

3.1. Quantification of Total Polyphenol, Flavonoid, and Anthocyanin Contents

3.2. Total Polyphenolic Content (TPC)

Polyphenolics are secondary metabolites popular found in fruits, mostly represented by flavonoids and phenolic acids. Their protective role against diseases in humans arouses great interest and the link between their intake and the prevention of some human diseases. Folin–Ciocalteu reagent was employed for the evaluation of total polyphenolic contents in FUS, FRS, FSP, and UFSP. Gallic acid was used as standard, its graph was drawn (r²: 0.994). The amount of total phenolic was determined from the standard graph equation as gallic acid equivalents per 1 mg of extract (GAE/mg extract). From Table 1, 16.85 ± 0.41, 10.535 ± 0.36, 9.55 ± 0.16, 11.525 ± 0.52 µg of gallic acid equivalents of polyphenolic content were estimated from 1 mg of FUS, FRS, FSP, and UFSP, respectively. With these values, it can be deduced that the high polyphenolic content is a significant influence on the antioxidant ability of green and red African berries. This study shows that the polyphenol compounds are a major factor in the antioxidant capacities of these berries.

Table 1

Quantification of Total Polyphenol, Flavonoid, and Anthocyanin Contents
Samples	Anthocyanin (mg/g)	Flavonoids (mg/g)	Polyphenols (mg/g)
FUS	2.652 ± 0.34	9.6 ± 0.61	16.85 ± 0.41
FRS	4.6005 ± 0.55	8.35 ± 0.34	10.535 ± 0.36
FSP	5.155 ± 0.23	7.5 ± 0.17	9.55 ± 0.16
UFSP	2.765 ± 0.12	9.1 ± 0.52	11.525 ± 0.52
FUS = Green Berries Skin, FRS = Red Berries Skin, FSP = Red Berries Seed Pulp, UFSP = Green Berries seed Pulp

3.3. Total Flavonoid Content (TFC)

The daily intake of fruits, vegetables, and foods containing flavonoids has been linked with protection against chronic diseases like heart disease and many types of cancer. Quercetin is a popular plant-derived flavonoid; flavonoids are an obvious group of polyphenolic compounds. They have been discovered to be renowned antioxidant agents since they are a peculiar and distinct group of polyphenols (Davis et al., 2009). The quercetin standard graph was plotted, and the total flavonoid content as quercetin equivalents was evaluated by the equation obtained from the standard graph. The results of FUS, FRS, FSP, and UFSP were found to be 9.6 ± 0.61, 8.35 ± 0.34, 7.5 ± 0.17, and 9.1 ± 0.52 mg/g, respectively (Table 1).

3.4. Total Anthocyanin Content (TAC)

Anthocyanins are an important class of secondary metabolites found mostly in fruits and vegetables; they are predominantly soluble in water phenolics and responsible for the colour formation, including blue, purple, pink, and red colours, in many medicinal plants and fruits (D’Archivio et al., 2007; Bravo, 1998). For example, anthocyanin glycosides, i.e., peonidin, petunidin, and malvidin, have been recognized as particular descriptors of wines’ colour (Boulton, 2001). From Table 1, total anthocyanin content is interestingly higher in FRS and FSP than in FUS and UFSP. While FRS and FSP have 4.6005 ± 0.55, 5.155 ± 0.23, FUS and UFSP have 2.652 ± 0.34, 2.765 ± 0.12, this obvious difference may have the result of cyanidin-3-O-glucoside presence in the former than the latter.

This study shows that total flavonoids, anthocyanins, and polyphenols contents varied broadly from different parts though these parts are from the same plant, grow, and were harvested on the same soil. The total flavonoids, anthocyanins, and polyphenol contents from the evaluated polyphenolic-rich extracts of these berries ranged from (mg REs/g extract) 7.1 ± 0.52 to 9.6 ± 0.61, 5.155 ± 0.23 to 2.652 ± 0.32, and 9.55 ± 0.16 to 16.85 ± 0.41. Significant differences in the quantity of biochemical were observed in different parts of the berries evaluated. The extract of the skin of the green berries possessed the highest total polyphenolic (mg GAEs/g extract) 16.85 ± 0.41 and total flavonoid (mg REs/g extract) 9.6 ± 0.61 contents. The extracts and seed plums of red berries possessed the highest anthocyanin content (mg REs/g extract): 4.6005 ± 0.55 and 5.155 ± 0.23. In the quantification of the total polyphenolic content in the four parts of these berry types, this assessment followed the following trend: FUS ˃ UFSP ˃ FRS ˃ FSP. A similar trend was noticed in flavonoid quantification (Table 1). Interestingly, the anthocyanin contents followed an opposite trend: FUS ˂ UFSP ˂ FRS ˂ FSP. This suggests that green berries possess higher polyphenolic contents when compared to deep red berries, though deep red berries possess higher anthocyanin contents when compared to green berries. Pale et al. (1998) quantified the anthocyanin content in wild berries of L. microcarpa and further discovered that the total anthocyanin content in the fruit was about 1300 mg/100g of the dry fruit skin (epicarp) of the deep red berries. Due to organoleptic interests, it has been suggested that secondary metabolites such as anthocyanins and proanthocyanidins possess a compelling effect on the color traits of fruits and food (Bueno et al., 2012). Hence, the high content of anthocyanins in the skin (FRS) and seed plum (FSP) of the red berries as compared to the green berries is not unfounded. Anthocyanins are found at fairly prominent concentrations in certain types of cereals, many roods and leafy vegetables (beans, onions, cabbage, aubergines) (Mazza and Maniati, 1993) But they have been reported to be abundant in fruits where the most common type is cyaniding, it has been found in red berries both skin and seed plum (Prakash and Sharma, 2014).

Table 2

**Antioxidant Activities of Green and Red Africa Berries**
Sample	FUS	FRS	FSP	UFSP	Ascorbic Acid
DPPH radical (mg TEs/g extract)	24.86 ± 0.92	22.23 ± 0.95	20.53 ± 0.21	56.26 ± 0.01	21.01 ± 0.47
ABTS radical cation (mg TEs/g extract)	81.24 ± 0.31	56.37 ± 0.71	72.03 ± 0.35	89.57 ± 0.73	101 ± 0.95
H₂O₂ reducing power (mg TEs/g extract)	56.05 ± 0.24	40.05 ± 0.11	31.64 ± 1.12	52.21 ± 0.12	53 ± 0.47
FUS = Green Berries Skin, FRS = Red Berries Skin, FSP = Red Berries Seed Pulp, UFSP = Green Berries Seed Pulp

Antioxidant Activity

Flavonoids and phenolics are renowned because of their antioxidant activity, though they are included in compounds tagged "polyphenols." They possess inherent significant properties to decrease the formation and production of free radicals, and also to scavenge free radicals’ toxicity with other dangerous reactive species such as hydroxyl radical (^•OH), nitric oxide (NO^•), peroxide radical (ROO^•) and superoxide radical (O^2•−) radicals (Pradeep & Sreerama, 2015; Oguntoye et al., 2018; Prior et al., 2012; Rice Evans et al., 1997). The total flavonoids, anthocyanins, and polyphenols contents with the antioxidant activity employing three assays i.e. DPPH radical, ABTS radical cation, and H₂O₂ reducing the power of polyphenolic rich extracts of FUS = Green Berries Skin, FRS = Red Berries Skin, FSP = Red Berries Seed Pulp, UFSP = Green Berries seed Pulp are presented in Tables 1 and 2. The antioxidant (in vitro) ability evaluation of these berries types was assessed (Table 2), the polyphenolic fraction of the seed pulp of the green berries displayed the highest activity in terms of DPPH radical scavenging (mg TEs/g extract) 56.26 ± 0.01, H₂O₂ (mg TEs/g extract) 52.21 ± 0.12 and maximum in ABTS (mg TEs/g extract) 89.57 ± 0.73. The polyphenolic fraction of the skin of green berries displayed the highest activity in terms of H₂O₂ reducing power (mg TEs/g extract): 56.05 ± 0.24; DPPH radical scavenging (mg TEs/g extract): 24.86 ± 0.92; and ABTS (mg TEs/g extract): 81.24 ± 0.31. The seed pulp of red berries showed the least activity in terms of both H₂O₂ reducing power (mg TEs/g extract) 31.64 ± 1.12 and DPPH radical (mg TEs/g extract) 20.53 ± 0.21 (Table 2).

No handy and adaptable method can assess the total antioxidant capacity of food, fruits, and others precisely and quantitatively (Koley et al., 2014). Two in vitro methods that are based on hydrogen atom transfer (HAT) and electron transfer (ET) were employed, i.e., DPPH and ABTS, while one in vitro method that is based on only electron transfer was employed, i.e., H₂O₂. In the DPPH assay, the antioxidant activity of the various parts of the two berry types followed the following trend: UFSP ˃ FUS ˃ FRS ˃ FSP and a similar trend was noticed in the ABTS assay too (Table 4.2). Interestingly, the H₂O₂ assay followed the following trend: FUS ˃ UFSP ˃ FRS ˃ FSP. This suggests that green berries possess higher antioxidant activity when compared to red berries. An important biologically active compound commonly found in vegetables, fruits, a wide variety of cereals, and foods that have aroused and held the interest of both consumers and researchers over the past two decades is polyphenols (Scalbert et al., 2005). They are aromatic phytocompounds universally spread throughout the plant kingdom, consisting of more than 7,900 substances with vastly distinct structural moieties. From small molecules with a molecular mass range (˂100 Da), i.e. phenolic acids, to big molecules of largely polymerized compounds (˃30000 Da) (Baby et al., 2018). The major motive for the interest in these classes of compounds is intertwined with the obvious recognition of their antioxidant activities, their abundance on our plates, and their apparent role in the prevention of many diseases associated with humans (Bach-Faig et al., 2011; Manach et al., 2004).

3.5. LCMS Result

The metabolome of four samples of Green and Red berries was analyzed and compared. A combination of both ionization modes (positive and negative) in MS full scan mode was applied for the molecular mass determination of the compounds in ethanolic extracts of the berries types. Compound identification was performed by comparing the observed m/z values and the fragmentation patterns with the literature. About 21 compounds shown in Tables 3–6 belong to different phenolic families, namely anthocyanidins, flavones, flavonols, flavan-3-ols, flavanones, hydroxycinnamic acids, hydroxybenzoic acids, and hydroxycoumarins.

Table 3

Compounds identified in Red Berries Skin
S/N	Identified compounds	Molecular formula	Calculated mass	Precursor ion, m/z [M-H]⁻ [M + H]⁺
1	Luteolin (Flavones)	C₁₅H₁₀O₆	286.2363	285.163
2	5-O-(4’-O-p-coumaroyl glucosyl) quinic acid (Hdroxycinnamic acids)	C₂₂H₂₈O₁₃	501		501.207
3	Hexose-hexose-N-acetyl	C₁₄H₂₅NO₁₀	366		367.569
4	Oleanoic acid (Terpenes)	C₃₀H₄₈O₃	457		457.278
5	Anmurcoic acid (Hydroxybenzoic acids)	C₃₀H₄₆O₅	487		487.258
6	Epicatechin (flavan-3-ols)	C₁₅H₁₄O₆	291		291.327
7	3-O-Methylgallic acid(Hydroxybenzoic acids)	C₈H₈O₅	185.0445	184.670
8	3-Feruloylquinic acid (Hydroxycinnamic acids)	C₁₇H₂₀O₉	367.1034		367.569
22	Cyanidin-3-O-glucoside (anthocyanins)	C₁₅H₁₁O₅

Table 4

Compounds identified in Red Berries Seed Pulp
S/N	Identified compound	Molecular formula	Calculated mass	Precursor ion, m/z [M-H]⁻ [M + H]⁺
9	L-Tryptophan	C₁₁H₁₂N₂O₂	204.225	204.283
10	Resveratrol (Stilbene)	C₁₄H₁₂O₃	229		229.191
11	Kaempferol (flavone)	C₁₅H₁₀O₆	285.04		285.361
12	6,7,3’,4’-tetrahydroxyisoflavone (flavone)	C₁₅H₁₀O₆	287.0550		287.771
13	3-O-Methylgallic acid (hydroxybenzoic acids)	C₈H₈O₅	185.04451	184.211
22	Cyanidin-3-O-glucoside	C₁₅H₁₁O₅

Table 5

Compounds identified in Green Berries Skin
S/N	Identified compound	Molecular formula	Calculated m/z	Precursor ion, m/z [M-H]⁻ {M + H]⁺
13	Quercetin (Flavones)	C₁₅H₁₀O₇	302.2357		303.093
14	Ellagic acid (Hydroxybenzoic acids)	C₁₄H₆O₈	302.1926		303.176
15	Hesperitin [Hesperetin] Flavanones	C₁₆H₁₄O₆	302.2788		303.252
6	Epicatechin (flavan-3-ols)	C₁₅H₁₄O₆	291		291.268
7	3-O-Methylgallic acid (hydroxybenzoic acids)	C₈H₈O₅	185.04451	184.41
5	Anmurcoic acid (hydroxybenzoic acids)	C₃₀H₄₆O₅	487		487.190
16	Equol	C₁₅H₁₄O₃	243.1016		243.217
17	Polydatin	C₂₀H₂₂O₈	389		389.424
18	Catechin gallate (flavan-3-ols)	C₂₂H₁₈O₁₀	441		441.395
19	Catechin (flavan-3-ols)	C₁₅H₁₄O₆	290.2681		291.822
20	Isorhamnetin 3-O-glucoside (Flavonols)	C₂₂H₂₂O₁₂	478.4029		479.307
21	Coumarin (Hydroxycoumarins)	C₉H₆O₂	147.0441	146.26
22	Chebulic acid	C₁₄H₁₂O₁₁	356.0365	355.39

Table 6

Compounds identified in Green Berries Seed Pulp
S/N	Identified compound	Molecular formula	Calculated m/z	Precursor ion, m/z
9	L-Tryptophan	C₁₁H₁₂N₂O₂	204.225	204.037
23	p-Coumaric acid isoprenyl ester (Hydroxycinnamic acids)		231	231.267
11	Kaempferol (Flavones)	C₁₅H₁₀O₆	287	287.282
17	Polydatin	C₂₀H₂₂O₈	389	389.289
24	Sinapic acid (Hydroxycinnamic acids)	C₁₁H₁₂O₅	225	225.198
25	Gallic acid (Hydroxybenzoic acids)	C₇H₆O₅	171	171.725

3.6. Phenolic acids

Phenolic acids are majorly divided into two classes: derivatives of hydroxycinnamic acid (hydroxycinnamic acid group) and benzoic acid derivatives (hydroxybenzoic acid group). This class of organic compounds is responsible for over 2.9% of over 300 dietary extractable polyphenol and phenolic compounds (Sharma, 2011). Hydroxybenzoic acids: Some of this class of secondary metabolites present in these berry-types are anmurcoic acid, 3-O-methylgallic acid, ellagic acid, and gallic acid (Fig. 1). The amount of hydrobenzoic acids present in edible plants is reportedly low, though Clifford and Scalbert (2000) discussed that in certain black radish, onions, and red fruits, their concentrations are up to several tens of milligrams per kilogram of fresh weight. They further highlight its excellent antioxidant activity and structural moiety (Clifford and Scalbert, 2000). Hydroxycinnamic acid: Some of this class of secondary metabolites present in these berry-types are 3-feruloylquinic acid, 5-O-(4’-O-p-coumaroyl glucosyl) quinic acid, p-coumaric acid isoprenyl ester, sinapic acid (Fig. 1). In contrast, hydroxycinnamic acids are more abundant than hydroxybenzoic acids in quite a few fruits and plants. The former occurs naturally in many medicinal plants as both trans and cis isomers, although the former is the most popular one, in the shikimate and phenylpropanoid pathways in plants (Kolaylı, 2010). Cinnamic acid plays an indispensable role, and shikimic acid is a precursor to many secondary metabolites present in herbs and shrubs, i.e., aromatic amino acids, alkaloids, and indole derivatives (Clifford and Scalbert, 2000). Hence, this group of organic compounds (hydroxycinnamic acids) plays a major role in the synthesis of other significant and essential compounds. Stilbenes and styrenes are some of the important compounds formed through decarboxylation reactions in nature by hydroxycinnamic acids (Sharma, 2011). Quinic and p-coumaric acids are reported to be present in all the parts of green and red berries; these classes of hydroxycinnamic acids and their derivatives represent more than 75 percent of the total hydroxycinnamic acids in a broad diversity of fruits. An aromatic ring attached with one or more hydroxyl (-OH) groups is the general character of phenolics, their structures are highly diverse i.e. from a simple molecule to that of a complex molecular mass (Balasundram et al., 2006). Oxidation reactions that are common in radical species are most disrupted by the phenolics by donating a hydrogen atom or chelating metals; hence, they proceed as reducing agents and antioxidants. Free radicals are changed to bland and harmless molecules by phenolics by donating electrons. Many pieces of research have shown the antioxidant properties of the phenolic contents of plants (Halliwell & Gutteridge, 1989; Köksal & Gülçin, 2008; Kumazawa et al., 2004; Bello et al., 2019a; Bello et al., 2019b; Bello et al., 2019c). This may account for the excellent antioxidant activity reported by the green and red African berries studied. Flavonols, flavones, flavanones, and flavan-3-ols: Some of these classes of secondary metabolites present in these berry types are quercetin, hesperitin (hesperetin), epicatechin, luteolin, kaempferol, 6, 7, 3’, 4'-tetrahydroxyisoflavone, catechin gallate, catechin, and isorhamnetin 3-O-glucoside. One of the largest groups of phytochemicals is called flavonoids. They consist of two phenyl rings joined together by three carbon atoms with an oxygen heterocycle ring; this moiety is mostly represented as C6-C3-C6. This group is renowned for its various biological activities (Köksal & Gülçin, 2008; Kumazawa et al., 2004).

The LC-MS-based analysis showed a high content of polyphenolics in the seed skin and seed pulp of the green and red wild Africa berries. The high content of total anthocyanins in the seed skin and seed pulp of the red berries revealed that anthocyanins may be responsible for the red colour of the fruit since total anthocyanin content is low in the green type. The LC-MS analysis was performed to obtain the phytochemical profile of these berry types, and about twenty-one (21) compounds belonging to different phenolic families, namely anthocyanidins, flavones, flavonols, flavan-3-ols, flavanones, hydroxycinnamic acids, hydroxybenzoic acids, and hydroxycoumarins, were identified. Quercetin is reportedly present in all the fractions. This study provided insight into the phytochemistry and pharmacological importance of these wild berries beyond their basic nutritional values. Thus, the high content of polyphenolics and anthocyanins in these fruit types may be of major interest to many industrial subdivisions, in addition to the development of functional foods, food supplements, pharmacological templates, and new products that will be very useful for human health.

Availability of data and materials

Not applicable

Competing interests

The authors declare there are no competing interests.

Funding

The authors receive no funding for this work

Authors' contributions

• Ogbesejana Abiodun B. analyzed the data, checked and prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

• Eveyln Ali Nok and Abdullateef Musharaf performed the experiments, prepared figures and/or tables, and approved the final draft of the manuscript.

• Bashir Musa performed the LC-MS analysis and interpret the data generated and approved the final draft of the manuscript.

• Bello Oluwasesan M. conceived and designed the experiments, and approved the final draft.

Acknowledgements

The authors wish to acknowledge Malam Dikko Illiayasu and Malam Kabir Safana, they went to look for the fruits in the bush.

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LC-MS Analysis, Total Polyphenol Content and Antioxidant Activity of Ethanolic Fractions of Skin (Epicarp) and Seed Pulp (Mesocarp) of Green and Red Wild Africa Berries.

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Abstract

Figures

1. Introduction

2. Material and Method

2.1. Collection

2.2 Extraction

2.3 Quantification

2.4 Total polyphenolic content

2.5 Total flavonoid content

2.6 Total Anthocyanins

2.7. Biological Activity

2.8. Antioxidant activity

2.9. 2-diphenyl-1-picrylhydrazil (DPPH) free radical scavenging assay

2.10. 2,2-azino-bis-(3-ethyl)benzothiazoline-6-sulfonic acid (ABTS) radical cation scavenging activity

2.11. Statistical analysis

2.12. Liquid Chromatography-Mass Spectrometry (LCMS)

3. Result and Discussion

3.1. Quantification of Total Polyphenol, Flavonoid, and Anthocyanin Contents

3.2. Total Polyphenolic Content (TPC)

3.3. Total Flavonoid Content (TFC)

3.4. Total Anthocyanin Content (TAC)

3.5. LCMS Result

3.6. Phenolic acids

4. Conclusion

Declarations

References

Status:

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