Introduction

Recently, metabolite profiling of medicinal plants is increasingly applied to assess the bioactive metabolites contributes to their nutritional and health value1. Sapotaceae is a family of flowering plants comprising about 1250 species of evergreen trees and shrubs distributed in 53 genera2. Several classes of phytochemicals including saponins, flavonoids and polyphenolic compounds, have been reported in Sapotaceae3. Moreover, different Sapotaceae species exhibit several biological activities such as antioxidant, anti-inflammatory, antibacterial, antiulcer, antidiabetic, and antifungal3. Among Sapotaceae genera, Mimusops is a tropical genus consisting of 57 species, many of which produce high-quality timber and edible fruit with significant nutritional and economic value. Owing to their bioactive properties including antioxidant, anti-inflammatory and antibacterial properties4, several species have been used to treat various ailments5.

Mimusops caffra E. Mey. ex A.DC, commonly known as Coastal Red Milkwood is a small to medium-sized fruit-bearing tree distributed in in KwaZulu-Natal, South Africa and the Former Transkei Region, Southern Africa6. Additionally, this tree forms up to 75% of the Coastal and Dune Forest in Mozambique7. M. caffra is also cultivated in Egypt at the Agricultural Research Center garden in Giza governorate, Egypt. The plant is well known for its commercial, ecological, and nutritional value6. The fruits of M. caffra are fleshy, bright orange-red when mature, edible and pleasantly sweet. The fruit pulp is rich in sucrose, glucose, and fructose with relatively low protein (5.65%) and lipid (6.76%) content. In the food industry, M. caffra fruit pulp is used for jelly and alcohol production. Traditionally, M. caffra extracts and decoctions have been widely used in ethnomedicine. In Zululand, South Africa, its bark extract is applied to treat wounds and sores, while bark maceration is used as an emetic. The root extracts are also used in the treatment of sexually transmitted infections such as gonorrhea. Additionally, M. caffra leaf extract has demonstrated anti-plasmodial and used to manage malaria8.

Recently, metabolomics tools have been widely applied for profiling of plant secondary metabolites9. The profiling of volatile metabolites in different plants of nutritional and economical value has been extensively reported to assess their quality10. Gas chromatography-mass spectrometry (GC-MS) is adopted for analysis of volatile compounds in different plant parts11,12. Volatile compounds are typically extracted by using either distillation and/or solvent extraction and most of them have been used widely for different biological activities13. n-Hexane is highly non-polar and volatile making it suited for extraction of non-polar volatile compounds while minimizing matrix interference in GC-MS analysis. Recently, dimethyl carbonate (DMC) solvent as an alternative to n-hexane for extracting volatiles due to its low eco-toxicity compared with n-hexane-extracted oil14. Unlike different GC methods, hyphenated technique such as ultra-high performance liquid chromatography (UHPLC) with Mass spectrometry is well adopted for profiling of non-volatile polar secondary metabolites1.

Despite the significant economic and nutritional values of M. caffra, studies on its phytochemical composition remain limited. Therefore, the main goal of this study is to profile the volatile and non-volatile secondary metabolites in M. caffra leaves by using GC-MS and LC-MS/MS analysis, respectively. To the best of our knowledge, this work presents the first comprehensive phytochemical profiling of M. caffra leaves. Additionally, the antioxidant and anti-inflammatory activities of M. caffra leaf methanol extract, n-butanol, and ethyl acetate extracts were evaluated using DPPH radical scavenging and NO inhibitory assays.

Results and discussion

Metabolites profiling of volatiles in M. caffra leaf via GC-MS analysis

The GC-MS analysis of M. caffra n-hexane extract led to the identification of 50 compounds (Fig. 1) belonging to various classes including monoterpene, aliphatic and aromatic hydrocarbons, alcohols, phenols, fatty acids/esters, and triterpenes (Table 1; Figs. 2 and 3). The GC-MS chromatogram, displaying the identified compounds and their corresponding peaks, is shown in Fig. 1. The phytoconstituents along with their retention time (RT) and concentration (peak area percentage), are presented in Table 1.

Fig. 1
Fig. 1
Full size image

GC-MS Chromatogram of M. caffra leaves n-hexane extract, 5: Decane 12: undecane, 22: Dodecane, 31: 2-phenyl undecane and 48: 24-Norursa-3,12-diene.

Table 1 Relative percentile of volatile metabolites detected in Mimusops caffra leaves hexane extracts analyzed using GC–MS.
Full size table
Fig. 2
Fig. 2
Full size image

Chemical composition of M. caffra leaves hexane extracts.

Fig. 3
Fig. 3
Full size image

Mimusops caffra tree and its leaves and pie charts of different volatile metabolites classes.

Aromatic hydrocarbons

Aromatic hydrocarbons were the predominant constituents detected in M. caffra n-hexane extracts, accounting for 41.5%, of the total composition. The most abundant compounds detected were 2-phenyl undecane (peak 31), 5-phenyl undecane (peak 28), and 2-phenyl dodecane (peak 36) were prevalent by 4.35, 4.12, and 3.47% respectively, Aromatic hydrocarbons and their phenylundecane derivatives have been reported for their notable antifungal and antibacterial activity15.

Aliphatic and monoterpene hydrocarbons

Aliphatic hydrocarbons constituted the second most abundant volatile class in the n-hexane extract of M. caffra, accounting for 26.7% of the total identified compounds. The most prevalent compounds were undecane (peak 12), dodecane (peak 22), and decane (peak 5) by 7.61, 6.31, and 3.4%, respectively. Undecane, a naturally occurring alkane hydrocarbon, has been reported to exhibit a potent anti-inflammatory and anti-allergic activities16. Dodecane has demonstrated antioxidant properties17. Unlike aliphatic hydrocarbons, monoterepene hydrocarbons were detected at lower percentage at 3.87% including 1-p-menthene (peak 2), 2-methyldecalin (peak 13), 1-ethyl-2,4-dimethylcyclohexane (peak 1), pentyl cyclohexane (peak 16) and 1-ethyl-2-propyl-cyclohexane (peak 11) were prevalent by 1.41, 0.69, 0.63, 0.61 and 0.53% respectively.

Triterpenes and phenols

Triterpenes constitutes 21.97% of the identified compounds in the n-hexane extract. Squalene (peak 44), β-amyrin (peak 47), 24-norursa-3,12-diene (peak 48), β-amyrin acetate (peak 49) and α-amyrin (peak 50) were the most abundant by 2.03, 4.24, 12.31, 1.53, and 1.86% respectively. Triterpenes α-amyrin and β-amyrin have been reported to have an antioxidant and a potential anti xanthine oxidase and tyrosinase enzyme inbibtors so used for preventing gout and skin hyperpigmentation respectively18 and they were identified in Manilkara zapota leaves of Sapotaceae family19. Squalene, a hydrophilic natural antioxidant, has demonstrated antiradical and antioxidant properties depending on the model system employed for the study20. Its presence in M. caffra is consistent with previous reported study on the petroleum ether extract of Mimusops elengi leaves, marking the first confirmation of squalene detection in M. caffra21. Phenols were also detected in the n-hexane extracts by α-tocopherol (peak 46) which accounted for 1.32%. α-Tocopherol is the major vitamin E compound found in leaf chloroplasts and has been reported to has a potential antioxidant activity22.

Organic acid ester

Organic acid esters accounted for 3.09% of the total identified compounds including oxalic acid, cyclohexylmethyl isohexyl ester (peak 21) and phthalic acid, bis(2-ethylhexyl) ester (peak 43) which accounted 0.29 and 2.8% respectively. Phthalic acid esters have been reported to function as allelochemicals, insecticidal, phytotoxic and have a potential antimicrobial activity23.

Alcohols and fatty acids/esters

Alcohols were detected in trace levels in the n-hexane extract of M. caffra leaves and represented by phytol (peak 15) 0.91%. Phytol is a diterpene member of the long-chain unsaturated acyclic alcohols, known for its roles as a natural antioxidant. While saturated aliphatic alcohols generally exhibit poor antioxidant activity, phytol exhibits a good antioxidant potential due to the allylic nature of its alcohol group24, and it has been identified in different plant species across different families25. Likewise, fatty acids/ester represented by palmitic acid, methyl ester (peak 42) 0.59% was detected at trace levels among volatile components of M. caffra leaves. It was previously identified in Manilkara zapota tree belonging to the Sapotaceae family26.

Secondary metabolites profiling of M. caffra leaf via UPLC-MS/MS analysis

UPLC-MS/MS analysis of M. caffra leaf crude methanol extract was performed in negative (Fig. 4A) and positive ionization modes (Fig. 4B). UPLC-MS/MS analysis led to the identification of 62 metabolites (Tables 2, 3) belonging to the various phytochemical classes including organic acids, phenolic acids, flavonoids in addition to their derivatives besides the presence of triterpenoids and fatty acids which were reported in negative ionization mode (Table 2). Moreover, phenolic acids, flavonoids were also reported in positive ionization mode beside to trace compounds belong to amino acids, alkaloids and sphingolipids with unknown compounds (Table 3). The order of elution of various chromatographic peaks occurred with decreasing polarity starting with organic acids, and simple phenolics, followed by flavonoid glycosides then aglycones and finally fatty acids and triterpenoids. Phenolic and flavonoid metabolites represented the most abundant class. Inspection of both negative and positive ionization modes revealed a higher detection level in the negative ionization mode especially for phenolic acids, and flavonoids. This is the first detailed metabolites characterization of M. caffra using high-resolution UPLC-MS. Therefore, the high content of glycosylated flavonoids and important bioactive phenolic compounds can contribute the value of M. caffra leaves as a potent antioxidant and promote beneficial effects on human health and well-being. However, further in vivo studies should be conducted with leaves to validate these effects.

Fig. 4
Fig. 4
Full size image

Base peak chromatogram of M. caffra leaf extract (A) analysis in negative mode (B) Positive mode.

Table 2 Chemical metabolites of M. caffra characterized by UPLC-Q-Exactive-MS/MS at negative mode.
Full size table
Table 3 Chemical metabolites of M. caffra characterized by UPLC-Q-extractive-MS/MS at positive mode.
Full size table

The identified metabolites are listed in Tables 2 and 3 which represents the negative and positive ionization mode, respresentively. Results revealed a total of 62 metabolites identified in the crude methanol extract, including organic acids (3 metabolites) which are identified in the negative mode only, phenolic acids and their derivatives (14 and 4 metabolites) in the negative mode and positive mode representively, flavonoids and flavonoidal glycosides (11 and 3 metabolites) in the negative mode and positive mode representively, as well as triterpenes (3 metabolites) and fatty acid derivatives (10 metabolites) which are identified in the negative mode only. amino acids, alkaloids and sphingolipids along with unknown compounds were reported in positive ionization mode only.

Organic acids

Organic acids and derivatives (compounds 1–3) were tentatively identified in M. caffra methanol extract, eluting early in the chromatogram among which malic acid (peak 1) (133.0137, C4H6O5), 2-furoic acid (peak 2) (111.0082, C5H4O3), and azelaic acid (peak 3) (187.0968, C9H16O4) were identified in the negative ionization mode and malic acid has already been widely reported in previous studies27. Organic acids belong to an important class of organic compounds that contribute to the flavor of fruits and vegetables. malic acid is one of the main organic acids responsible for the flavor notes of most fruits28.

Phenolic acids and its derivatives

Phenolic compounds represent a diverse class of organic compounds characterized by their aromatic nature and the presence of one or more hydroxyl groups attached to the phenyl ring29. These compounds have garnered significant attention due to their various physiological and pharmacological properties. Phenolics are widely distributed throughout the plant kingdom and serve as powerful antioxidants and exhibiting potential benefits for human health30. M. caffra leaves have been identified to contain a diverse array of phenolic compounds such as gallic acid (C7H6O5) in peaks 4 and 48 at m/z (169.0135 and 171.0287) which has been identified according to31,32. Gallic acid, one of the hydroxybenzoic acids, was reported in previous studies as a polyphenolic antioxidant in fruits of Pouteria species belongs to Sapotaceae family33. Among phenolic acids, gallic acid is tremendously well absorbed into the human body, compared with other polyphenols. It was shown to have a positive effect against cancer cells under in vitro conditions34. In addition, quinic acid (C7H12O6) in peaks 7 and 49 at m/z (191.0551 and 193.0707) has been identified as follows32,35. It was previously reported in methanol extract of Mimusops elengi with [M-H]− at m/z 191.056436, confirming quinic acid detection in caffra species which was reported for the first time. Moreover, p-coumaroylquinic acid (C16H18O8) in peaks 10 and 51 at m/z (337.0915 and 339.1072) has been identified according to32,37. The previous phenolic compounds have been identified in the negative and positive ionization modes respectively. Some of the phenolic compounds present in the leaves (compounds 12–17) were found to be linked to one or more sugar residues where they are identified in the negative ionization mode only such as vanillic acid hexoside, caffeic acid-O-hexoside, syringic acid-O-hexoside in peaks 13, 15, and 16 with [M-H]− at m/z (329.0895, C14H18O9-) with MS2 fragments at m/z 329, ( 341.0896, C15H18O9-) with MS2 fragments at m/z179 and135 and (359.0999, C15H20O10-) with MS2 fragments at m/z 197, 182, 167, 153, 138, 123, 95 respectively, vanillic acid-O-hexoside has been identified according to38. While caffeic acid-O-hexoside and syringic acid-O-hexoside according to39. Vanillic acid-O-hexoside and syringic acid-O-hexoside have been reported in previous studies to have antioxidant activity40.

Flavonoids and its derivatives

Flavonoids are a group of natural polyphenolic compounds consist of a flavan nucleus composed of two benzene rings linked by a heterocyclic pyran ring or pyrone, they have a powerful antioxidant and anti-inflammatory activities41. The subclasses of flavonoids commonly found in plants include flavones, flavanones, flavonols, and isoflavones, A total of 14 flavonidal compounds identified in both negative and positive mode (11 and 3), respectively. The identified free flavonoids including quercetin, epicatechin, gallocatechin and ethyl 2,4 dihydroxy-3-(3,4,5-trihydroxybenzoyl)oxybenzoate were determined in this study in peaks (18–21) with [M-H]− at m/z ( 301.0341, C15H10 O7-), (289.0705, C15H14O6_), (305.0654, C15H14O7-) and (349.0581, C16H14O9_) and exhibiting MS2 fragments at m/z 151 and 17935, 245.0794, 203.0661, 151.0388, 109.030035, 305, 261, 219, 179, 12542 and 198.0759,197.0420, 169.0096, 124.014943 respectively. Epicatechin has shown different pharmacological activities such as antiviral44 and antioxidant45 activities. Recently, epicatechin has been reported to has a potential use in the management of obesity and periodontitis46. Moreover, peaks (52–54) displayed [M + H]+ at m/z (287.055, C15H10O6+) with MS2 fragments at m/z 265 and 275,( 303.05, C15H10O7+ ) with MS2 fragments at m/z 273 and 289 and (291.0863, C15H14O6+) with MS2 fragments at m/z 262 were assigned as 5, 7, 20, 30—tetrahydroxyflavone, 3,5,7,20,50—pentahydroxyflavone and ent-fisetinidol-4-β-ol, respectively according to32. Some of the flavonoidal compounds present in the leaves (compounds 22–28) in the negative ionization mode were found to be linked to one or more sugar residues such as galloyl derivatives of glucose in peaks 22 and 23 [M-H]− which were annotated as galloyl hexoside (m/z 331.0682, C13H16O10−) with MS2 fragments at m/z 331,169,151, 12542 and ethyl-O-β-D-(6’-O-galloyl)- glucopyranoside (m/z 359.0999, C15H20O10−) with MS2 fragments at m/z 169.014043, respectively. Additionally, five O-type flavonoid glycosides (compounds 24–28) were identified comprising apigenin-7-O-β-D-glucoside (m/z 431.0967, C21H20O10−) and along with fragment peak at 269.0439 based on result analysis from35. Additionally, the other O-type flavonoid glycosides were detected according to47 assigned as kaempferol-3-O-deoxyhexoside (m/z 431.0967, C21H20O10−) with MS2 fragments at m/z 431.1002, 285.0408, 284.0332, 255.0303, 227.0352 and 229.0506, kaempferol-3-O-hexoside (m/z 447.0917, C21H20O11−) with MS2 fragments at m/z 447.0945, 285.0398, 284.0318, 255.0292, 227.0340 and 151.0031, quercetin-3-O-hexoside (m/z 463.0868, C21H20O12−) with MS2 fragments at m/z 463.0899, 301.0355, 300.0271, 271.0247, 255.0295, 243.0296, 178.9985 and 151.0035 and myricetin-3-O-hexoside (m/z 479.0808, C21H20O13−) with MS2 fragments at m/z. 479.0841, 317.0286, 316.0221, 287.0193 and 271.0239 respectively. Quercetin-3-O-hexoside has been detected in several previous studies and has been reported as a potent antioxidant flavonidal compound48. In this study it was identified and detected for the first time in M. caffra leaf according to47.

Nitrogenous compounds

Nitrogen-containing metabolites were detected though at lower levels, among which l-tryptophan and gentiatibetine were characterized in this study (peak 56), (peak 55) respectively. [M+H]+ at m/z (205.0971, C11H12N2O2+) with MS2 fragments at m/z 188.0708, 146.0603 and 118.0654 was annotated as L-tryptophan based on result analysis from49. While gentiatibetine with [M+H]+ at m/z (166.0861, C9H11NO2+) exhibiting MS2 fragments at m/z 143,151 according to32.

Sphingolipids

Sphingolipids are a class of lipids with high structural diversity and biological pleiotropy. Three sphingolipid components were determined including dehydrophytosphingosine, octadecasphinganine and phytosphingosine by LC-MS/MS method Peaks 57, 58, and 59 exhibited molecular ions [M + H]+ at m/z 316.2843, 302.305 and 318.3 respectively, Most of the sphingolipids and their dihydro equivalents fragment to backbone ions with m/z 264 in positive ion mode as a key for the identification of sphingolipids in Manilkara zapota fruit50. Most notably, fragment ions (m/z 281, 280) are for dehydrophytosphingosine, whereas fragment ions at m/z 282, 264 correspond to phytosphingosine, moreover, fragment ions at m/z 285,284 are for octadecasphinganine. These metabolites are reported here for the first time in caffra leaf, and likely to account for a wide array of therapeutic indications such as treatment of cancer, inflammations, and metabolic disorders51.

In vitro antioxidant activity assays via DPPH free radical scavenging activity

The DPPH assay is a rapid and efficient method for evaluating free radical scavenging activity29, as it measures the ability of an extract to donate an electron or hydrogen radical to stabilize free radicals. In the present study, the antioxidant activity of M. caffra leaf extracts was assessed by measuring their percentage inhibition of DPPH radicals, as listed in Table S1. A total of eleven varying concentrations (0, 2.5, 5, 10, 20, 40, 80, 160, 320, 640, and 1280 µg/ml) of different solvent extract of M. caffra demonstrated different percentage of inhibition. The results demonstrated a concentration-dependent increase in scavenging activity across all extracts, with the highest inhibition observed at 1280 µg/ml. Among them, the crude methanol extract showed the highest scavenging activity (98.52%), followed by ethyl acetate (97.13%) and n-butanol fraction (94.38%). The antioxidant activity measurement has revealed that the crude methanol extract of M. caffra demonstrated greater antioxidant activity than ascorbic acid (Fig. 5A–D). previous reports have shown that methanol extract of medicinal plants possessed good pharmaceutical activity. Recent finding has reported that methanol extract of M. caffra leaves potentially have a free radical scavenging activity. Ethyl acetate extract has been used for extracting some phenolic and nitrogenous compounds. These compounds are known to scavange the free radicals and reactive oxygen species (ROS) including superoxide anion, hydroxyl radicals and singlet oxygen. Ethyl acetate extract of M. caffra showed a significant dose-dependent inhibition of DPPH activity. Among the two previous solvent extracts, crude methanol extract of M. caffra leaves exhibited highest potential antioxidant in a concentration dependent manner.

Fig. 5
Fig. 5
Full size image

The radical DPPH scavenging activity, represented by percentage of inhibition, of the three different solvent extract compared to ascorbic acid. (A) Percentage of inhibition of total extract; (B) percentage of inhibition of n-butanol extract; (C) percentage of inhibition of ethyl acetate extract and finally; (D) percentage of inhibition of ascorbic acid.

The IC50 value was calculated to determine the concentration of the sample required to inhibit 50% of free radical. The lower the IC50 value, the higher the antioxidant activity of samples. The observed IC50 value confirm that the crude methanol extract exhibited highest antioxidant activity followed by ethyl acetate extract and n-butanol extract, respectively (Table 4). Interestingly, the IC50 value of the crude methanol extract was also lower than ascorbic acid. According to52, extracts which possess IC50 values ranging from 50 to 100 mg/mL is considered to exhibit intermediate antioxidant activity. Meanwhile, extracts with IC50 value ranging between 10 and 50 mg/mL is considered to possess strong antioxidant activity (Table S3). In this case, the crude methanol extract, ethyl acetate and n-hexane extraxts possessed strong antioxidant activity.

Table 4 IC50 value of DPPH radical scavenging activity.
Full size table

Compared to other previously reported studies, Mimusops elengi Linn. (one of the most important species belonging to the Sapotaceae family) was reported for its strong antoxidant activity due to its high phenolic content53. The methanol extract of M. elengi bark showed marked inhibition 90.61% at a dose of 20 µg/mL and ascorbic acid as a reference compound showed marked inhibition 93.15%. Additionally, the DPPH assay was conducted with M. caffra, M. Zeyheri, M. kummel, and M. laurifolia at a concentration of 20 µg/ml5, revealing that the hydro-methanolic extracts exhibited scavenging effect of 67%, 56%, 42%, and 31%, respectively. The calculated IC50 value revealed that M. caffra was the best antioxidant among the previous species. A recent finding shows a marked inhibition 80.61% for the methanol extract of M. caffra leaf and 76.31% for standard ascorbic acid at the same dose. Therefore, the methanol extract of M. caffra leaf showed strong antioxidant activity by inhibiting DPPH radical scavenging activities when compared with standard ascorbic acid. Such activity of M. caffra owing to its richness in phenolic and flavonoid compounds which play a pivotal role in the antioxidant capacity. Although the antioxidant activities found in vitro experiment were only indicative of the potential health benefit, these results remain important as the first step in screening antioxidant activity of M. caffra leaf. Thus, it can be concluded that methanol extract of M. caffra leaf can be used as an accessible source of natural antioxidants with consequent health benefits.

In vitro anti-inflammatory activity via NO inhibitory effect activity

Nitric oxide (NO) is a pro-inflammatory mediator that plays a key role in the pathogenesis of inflammatory disorders, particularly when produced in excess underabnormal situations54. NO is synthesized and released into the endothelial cells by the help of nitric oxide synthases (NOSs), which convert arginine into citrulline generating NO in the process. NO has been recognized for its role invasodilatation in cardiovascular system. Additionally, it participates in immune responses through cytokine-activated macrophages, which release NO in high concentrations55.

Abnormal NO production is often associated with various animal and human diseases. In some cases, preventing a decrease in constitutive NO production in the vasculature may mitigate the development of vascular disease, while inhibition of uncontrolled NO production could also serve as a therapeutic target56. Although NO and other free radicals are generated in our body during inflammation for specific metabolic purposes, they also involved in regulation of cell growth, energy production and intercellular signaling. Thus, when an imbalance between free radical generation and body defence mechanisms occurs, free radicals can attack proteins in tissues, lipids in cell membranes, DNA and enzymes inducing oxidations, which cause protein modifications, membrane damage and DNA damage leading to inflammation and other series of human illnesses such as heart diseases and cancer57. Therefore, NO inhibitors represent important therapeutic advance in the management of inflammatory diseases, flavonoids were previously reported to have a markedly decline in NO production at higher doses as compared to control58. In the present study, there are many examples of flavonoids detected from the crude methanol extract of M. caffra leaf with anti-inflammatory activity (Tables 2, 3), such as quercetin59. Additionally, polyphenolic, proanthocyanidin, alkaloid, terpenoid and steroid compounds are usually responsible for the anti-inflammatory activities of plant extracts. These secondary metabolites act on different targets involved in the inflammatory pathway57. Among the phenolic content that have been detected in M. caffra leaf, gallic acid has received increasing attention for its powerful anti-inflammatory properties60.

In our study nitric oxide scavenging activity was evaluated using crude methanol extract, ethyl acetate and n-butanol fractions. The reductive potential of all three extracts exhibited dose dependent activity, as shown in Fig. 6. The IC50 values were calculated for all three extracts, Fig. 6A–C corresponding to 137.9 ± 2.76, 419.3 ± 7.89 and 289.6 ± 4.08 µg/ml. respectively. The results are summerized in Table S2 and graphically represented in Fig. 6. The regression analysis revealed a linear increase in % scavenging activity with increasing extract concentration for all three extracts (Fig. 6). These findings confirm that the the crude methanol extract showed potent anti-inflammatory activity compared to the ethyl acetate and n-butanol fractions. All three extracts showed good anti-inflammatory activity in relation to their total flavonoid and phenolic content.

Fig. 6
Fig. 6
Full size image

The radical NO scavenging activity, represented by percentage of inhibition, of the three different solvent extract compared to ascorbic acid. (A) Percentage of inhibition of total extract; (B) percentage of inhibition of n-butanol; and (C) percentage of inhibition of ethyl acetate extract.

Conclusion

This study presents a comprehensive phytochemical profiling of M. caffra leaf extracts, targeting both volatile and non-volatile secondary metabolites using GC-MS and UPLC-MS/MS analyses. In vitro antioxidant and anti-inflammatory activity were assessed for the crude methanol extract, ethyl acetate and n-butanol fractions of M. caffra leaves by DPPH free radical scavenging and nitric oxide inhibition assays, respectively. A total of 50 volatile metabolites and 62 secondary metabolites were identified via GC-MS and UPLC-MS/MS, respectively, revealing a diverse range of bioactive constituents in the leaf extract. Notably, Phenolic and flavonoid compounds constituted about 32 compounds form the identified secondary metabolites by using UPLC-MS/MS analysis highlighting their abundance in M. caffra leaves. The DPPH radical scavenging assay and NO inhibition assay revealed that the crude methanol extract exhibited potential antioxidant with IC50 of 9 µg/ml and anti-inflammatory activity with IC50 of 137 µg/ml. These bioactivities are attributed to the richness with phenolic and flavonoid compounds detected via UPLC-MS/MS. The results of this study indicates that M. caffra crude methanol extract holds significant potential as a therapeutic agent for preventing or slowing aging and oxidative stress-related degenerative diseases. Additionally, ethyl acetate and n-butanol fractions are also exhibited good antioxidant activity, further supporting M. caffra as a valuable natural source of antioxidant. Further isolation and purification of specific bioactive metabolites from M. caffra leaves is recommended for future studies along with more indepth biological investigation. Moreover, green extraction techniques is recommended to be used instead of conventional solvent extraction such as electrochemical extraction, ultrasonic-assisted extraction, deep eutectic solvents, ionic liquids, enzyme-assisted extraction, microwave-assisted extraction and subcritical water extraction. In summary, M. caffra leaves represents a promising natural source of antioxidants and anti-inflammatory agents, with potential for development into the health-promoting dietary supplements.

Materials and methods

Plant material

Mimusops caffra E. Mey. ex A.DC leaf was collected from a tree growing in the Agricultural Research Center garden in Giza governorate (30.0209° N, 31.2113° E), Egypt, during in December 2020. The plant was botanically identified by Prof. Dr. Reem Samir Hamdy, Professor of Plant Taxonomy, Botany Department, Faculty of Science, Cairo University. Avoucher specimen was deposited in Pharmacognosy Department Herbarium, Faculty of Pharmacy, Egyptian Russian University under code (MCL1/21). About 10 Kg of M. caffra fresh leaves were washed with tap water and dried under shade.

Plant extraction

The air-dried ground M. caffra leaves (100 g) were extracted with n-hexane as a nonpolar solvent which used later for study of volatile oil profile using GC-MS method. About 1400 g of M. caffra powder was subjected to cold maceration process in a conical flask using 100% methanol for 3 days at a room temperature. The extract was filtered using double ring 18.0 cm filter paper and the extraction process was repeated three times. The combined extracts were concentrated under reduced pressure at 55 °C by rotary evaporator in order to obtain the crude methanol extract (135 g). About 115 g of the crude extract were fractionated in separating funnel using solvents with different polarity as ethyl acetate, n-butanol, petrolium ether, and chloroform and each extract was prepared into final concentrations to yield 7 g, 18.5 g, 5 g and 7.5 g, respectively. The crude methanol extract, ethyl acetate, and n-butanol fractions were used subsequently in the investigation of antioxidant and anti-inflammatory activities.

GC-MS analysis of volatiles in M. caffra leaf

Gas chromatography analysis was performed at Pharmacognosy Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt on April 2021. M. caffra n-hexane extract was subjected to GC-MS analysis. n-Hexane is highly non-polar and volatile making it suited for extraction of non-polar volatile compounds while minimizing matrix interference in GC-MS analysis61. Mass spectra were recorded using Shimadzu GCMS-QP2010 (Koyoto, Japan) equipped with an Rtx-5MS fused bonded column (30 m × 0.25 mm i.d. X 0.25 μm film thickness) (Restek, USA) equipped with a split − splitless injector (1.0 µL of the prepared n-hexane extract was injected). The initial column temperature was kept at 50 °C for 3 min (isothermal) and programmed to 300 °C at a rate of 5 °C/min and kept constant at 300 °C for 10 min (isothermal). The injector temperature was 280 °C. The helium carrier gas flow rate was 1.37 mL/min. All the mass spectra were recorded by applying the following condition: (equipment current) filament emission current, 60 mA; ionization voltage, 70 eV; and ion source, 220 °C. Diluted samples (1% v/v) were injected with the split mode (split ratio 1: 15). Identification of volatile metabolites composition was performed by comparing their retention indices in relation to n-alkanes (C6 − C20), mass matching to NIST17, Wiley library database. Peaks were first deconvoluted using AMDIS software10.

High-resolution ultra high-performance liquid chromatography analysis (UPLC-MS/MS)

About 100 mg of the crude methanol extract was dissolved in 5 mL 100% methanol and 3 µl was subjected to chromatographic separation using an I-Class UPLC system. The UHPLC analysis was performed on an Acquity UHPLC System (Waters) equipped with a HSS T3 column (100 × 1.0 mm, particle size 1.8 mm; Waters). The analysis was carried out by applying the following binary gradient at a flow rate of 150 mL min− 1: 0–1 min, isocratic 95% A (water/formic acid, 99.9/0.1 [v/v]), 5% B (acetonitrile/formic acid, 99.9/0.1 [v/v]); 1–16 min, linear from 5 to 95% B; 16–18 min, isocratic 95% B; and 18–20 min, isocratic 5% B. The injection volume was 3.1 mL (full loop injection). Eluted compounds were detected from m/z 90 to 1000 using a MicroTOF-Q hybrid quadrupole time-of flight mass spectrometer (Bruker Daltonics) equipped with an ApolloII electrospray ion source in negative and positive (deviating values in brackets) ion modes using the following instrument settings: nebulizer gas, nitrogen, 1.4 (1.6 bar); dry gas, nitrogen, 6.l min− 1, 190 °C; capillary, -5000 V (+ 4000 V); end plate offset, 500 V; funnel 1 RF, 200 Vpp; funnel 2 RF, 200 Vpp; in-source CID energy, 0 V; hexapole RF, 100 Vpp; quadrupole ion energy, 5 eV (3 eV); collision gas, argon; collision energy, 7 eV (3 eV); collision RF, stepping 150/350 Vpp (200/300 Vpp), (timing 50/50); transfer time, 58.3 µs; prepulse storage, 5 µs; pulser frequency, 10 kHz; and spectra rate, 3 Hz. Internal mass calibration of each analysis was performed by infusion of 20 µL 10 mM lithium formate in isopropanol : water, 1:1 (v/v), at a gradient time of 18 min using a diverter valve. For auto-MS/MS analysis, precursor ions were selected in Q1 with an isolation width of ± 3–10 Da and fragmented at collision energies of 15–70 eV using argon as a collision gas. Product ions detection was performed using the same settings as above, but with funnel 2 RF 300 Vpp in negative mode. Metabolites were characterized by their UV-vis spectra (210–650 nm), retention times relative to external standards, accurate MS and the domino MS/MS spectra in comparison to our in-house database, phytochemical dictionary of natural products database and reference literature.

Antioxidant activity via DPPH radical scavenging activity

The antioxidant activity of extract was performed at the Regional Center for Mycology and Biotechnology (RCMB) at Al- Azhar University by using the DPPH free radical scavenging assay in triplicate and average values were considered29. Freshly prepared (0.004%w/v) methanol solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical was prepared and stored at 10 °C in the dark. A methanol solution of the test sample was prepared. A 40 uL aliquot of the methanol solution was added to 3 ml of DPPH solution. Absorbance measurements were recorded immediately with a UV-visible spectrophotometer (Milton Roy, Spectronic 1201). The decrease in absorbance at 515 nm was determined continuously, with data being recorded at 1 min intervals until the absorbance stabilized (16 min). The absorbance of the DPPH radical without antioxidant (control) and the reference compound ascorbic acid were also measured. All the determinations were performed in three replicates and averaged. The percentage inhibition (PI) of the DPPH radical was calculated according to the formula:

$${\text{PI }}={\text{ }}\left[ {\left\{ {\left( {{\text{AC}} - {\text{ AT}}} \right)/{\text{ AC}}} \right\}{\text{ }} \times {\text{ 1}}00} \right]$$
(1)

Where AC = Absorbance of the control at t = 0 min and AT = absorbance of the sample + DPPH at t = 16 min62.

The 50% inhibitory concentration (IC50), the concentration required to inhibit DPPH radical by 50%, was estimated from graphic plots of the dose response curve.

Anti-inflammatory activity via nitric oxide (NO) inhibition activity

NO radical inhibition activity of the tested samples was determined according to method of Marcocci et al.63 by using a sodium nitroprusside (SNP). NO radical generated from SNP in aqueous solution at physiological pH reacts with oxygen to produce nitrite ions that were measured by the Greiss reagent. The reaction mixture (2 mL) containing various concentrations of the tested samples and SNP (10 mM) in phosphate buffered saline (PBS; pH 7.4) was incubated at 25 °C for 150 min. At the end of the incubation period, 1 mL of reaction mixture samples was diluted with 1 mL Greiss reagent (1% sulphanilamide (w/v) in 5% phosphoric acid (v/v) and 0.1% naphthyl ethylene diamine dihydrochloride). The mixture was incubated at 25 °C for further 30 min. The absorbance of these solutions was measured at 546 nm against the corresponding blank solution (without sodium nitroprusside). All the tests were performed in triplicate. The percent inhibition activity was calculated using the formula :

$$\:\text{I}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{n}\:\text{\%}\:=\left[\frac{\text{A}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}-\text{A}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}{\text{A}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\right]\times\:100$$
(2)

where, A control is the absorbance of the control reaction at 546 nm and Atest represents the absorbance of a test reaction at the same wavelength. Tested material concentration providing 50% inhibition (IC50) was calculated from the graph plotting inhibition percentage against concentration64.

Statistical analysis

The results of biological investigation were analyzed in triplicate and displayed as average ± standard deviation of the mean (SD) (Tables S1 and S2). By using the t-tests analysis comparing each extract to ascorbic acid in DPPH assay significant differences (p < 0.05) appear at lower concentrations (≤ 80 µg/ml), where ascorbic acid retains higher activity. In NO inhibition assay, significant differences (p < 0.05) were observed at all concentrations, and crude methanol extract had significantly higher NO inhibition.