Chemical composition, antioxidant and antimicrobial activities of Himalayan Fraxinus micrantha Lingelsh leaf extract

Abstract The present investigation determines the phytochemical analysis, antioxidant and antibacterial potential of methanol extract of Himalayan Fraxinus micrantha (Lingelsh) leaves. Chemical analysis of extract revealed the presence of fifty five compounds. The extract was strongly characterised by tyrosol, esculetin, β-sitosterin, l-(+)-ascorbic acid 2,6-dihexadecanoate, (Z,Z)-6,9-cis-3,4-epoxy nonadecadiene as main components (62.6%). The F. micrantha extract presents IC50, 43.93 µg/ml and 68.90 µg/ml using DPPH and H2O2 scavenging assay, respectively. The antibacterial activity of F. micrantha was evaluated for five microorganisms showed that the extract had a remarkable inhibitory potential with a mean zone diameter of inhibition ranging from 11 to 22 mm. The MIC of the extracts ranged from 7.8 to 250 µg/ml and MBC value range 31.25 to 125 µg/ml. F. micrantha showed significant antibacterial activity against B. subtilis (22 mm, ZOI) with MIC and MBC values 7.8 and 31.25 µg/ml respectively. Graphical Abstract


Introduction
Fraxinus micrantha, Lingelsh (Vernacular name-Angu, English name-Ash and family-Oleaceae) is a temperate Himalayan multipurpose deciduous tree species of high medicinal value and ethnobotanical importance (Iqbal 2008). Fraxinus species have been used in folklore medicine for their diuretic and purgative effects due to presence of several glycosides including fraxin, acoumarin glycoside, which is an active diuretic agent. The leaves and the bark are also used for the treatment of constipation, arthritis, rheumatic pain, cystitis and itching scalp (Iqbal 2008).In recent years the increased interest in the phytochemistry of Fraxinus is motivated by the discovery of the secoiridoid glucosides that constitute major metabolites in the genus of family Oleaceae (Kostova and Iossifova 2007).
To the best of our knowledge, the chemical composition of F. micrantha has been poorly documented and there is no published report in terms of its phytochemistry and its biological and pharmacological activities. Therefore, the present study was carried out to evaluate Himalaya F. micrantha, as a source of natural antioxidant and antimicrobial agent.

Chemical composition of extract
The results of present investigation demonstrated that the calculated yield was 12.72% for F. micrantha extract (Supplementary material Table S2). Based on the chemical analysis of methanol extract, 55 components were identified; among these five major compounds were represented 62.6% of the total detected constituents. Supplementary material Table S1, represented that the major constituents of the analysed extract were (Z,Z)-6,9-cis-3,4-epoxy nonadecadiene (22.35%); tyrosol (15.2%); b-sitosterin (10.15%); l-(þ)-ascorbic acid 2,6-dihexadecanoate (8.96%); esculetin (5.96%).The structures of all identified five major compounds are shown in Supplementary material Figure S1. The occurrence of secoridoids i.e. tyrosol is a characteristic feature of Fraxinus species which is biological active phenylethanoids compound (Kostova and Iossifova 2007).The coumarins are found in a 20 free forms or as glucosides in Fraxinus species so far (Hegnauer 1969;Murray et al. 1982). Joshi et al. (1998) reported two coumarins glucosides, viz., esculetin and isoscopoletin in essential oil of F. micrantha. Present phytochemical analysis also supported the earlier investigations for presence of tyrosol, coumarins as major compounds in Fraxinus species.

Antioxidant activity
As evident from the Supplementary material Table S3, the results of the antioxidant activities (scavenging free radical DPPH, H 2 O 2 and Fe þ3 ) showed that the tested methanol extract of F. micrantha has high antioxidant potential; IC 50 is 43.93 mg/ml for DPPH assay and 68.90 mg/ml for H 2 O 2 assay. These capacities of tested extract were less than ascorbic acid (19.84 and 12.56 mg/ml for DPPH and H 2 O 2 assay respectively). From the Supplementary material Table S3, it can be seen that mean of IC 50 values of F. micrantha extract for DPPH radical and used ascorbic acid (positive control) were positively correlated with each other whereas mean of IC 50 values of tested extract and used ascorbic acid have negative correlation for hydrogen peroxide assay.
Some earlier workers have investigated Fraxinus sp. for their antioxidant potential as F. mandshurica and F. rhychophlla by Vasilevskaya et al. (1994), F. excelsior by Meyer et al. (1995) and Schempp et al. (2000), F. angustifolia by Fale et al. (2013), F. floribunda by Subba and Mandal (2015) whereas the present study on antioxidant potential of Himalayan F. micrantha leaves is the first report. This strong antioxidant potential of F. micrantha extract might be due to presence of high content of tyrosol (Angiolella et al. 2018), which is most active antioxidant compound after gallic acid (Bernini et al. 2015).The efficiency of studied plant extract to reduce Fe þ3 to Fe þ2 as an antioxidant agent presented in Supplementary material Table S2, which was expressed in AAE/g of dry extract. Obtained data showed that methanol extract has significant ferric reducing antioxidant power (38.27 ± 0.62 AAE/g of dry extract), which indicated the presence of some compounds that are electron donors and could react with free radicals to convert them into more stable products.

Antibacterial activity
According to the results in Supplementary material Table S4, the extract obtained from F. micrantha had remarkable inhibitory effect against the growth of most microorganisms tested except Gram-negative bacteria: X. phaseoli at 1000 mg/ml concentration. The results indicated that Gram-positive bacteria: B. subtilis is most sensitive for methanol extract (1000 mg/ml) with the largest inhibition zone, 22 mm and lowest MIC and MBC values (7.8 and 31.25 mg/ml respectively) which was comparable to standard drugs gentamycin and kanamycin (ZOI, 23 mm), Supplementary material Table S4. Antibacterial results of present study also showed that tested plant extract had effective inhibition against E. chrysanthemi with zone of inhibition 19 mm at 1000 mg/ml as compare to used standard drug, kanamycin (ZOI, 16 mm). This significant antibacterial inhibition of F. micrantha extract might be due to presence of high content of antibacterial compound, (Z,Z)-6,9-cis-3,4-epoxy nonadecadiene in tested extract (Supplementary material Table S1), which is individually or synergistically inhibited the growth of the pathogenic bacteria (Patra et al. 2015). Present study also suggested that, tyrosol (phenolic compound) was a major compoundin F. micrantha leaves, recent investigations reported that tyrosol acts as an antibacterial agent against Grampositive and Gram-negative bacteria (Tafesh et al. 2011;Martinez et al. 2018).) The biologically active coumarins compound 'esculetin; was also detected in the leaves of F. micrantha. De Souza et al. (2005) reported that esculetin displayed fairly high antibacterial activity in relation to Gram-positive bacteria (MIC, 500 mg/ml), which is probably due to the facilitated interaction with the peptidoglycan found in the cell wall.The Gram-negative bacteria species included in the study (E. coli, A. tumefaciens and X. phaseoli) were less susceptible to extract antibacterial activity. It is well documented that Gram-negative microorganisms are intrinsically resistant to a range of compounds with antibacterial activity due to the presence of the outer membrane (Lima et al. 2018;Ghai and Ghai 2018).
Let us assume that the mean activity of F. micrantha leaf methanol extract is less than used standard drugs (kanamycin and gentamycin) against tested bacterial strains. By applying student's t-test, it can be estimated that the negative correlations were found between mean activities of kanamycin, gentamycin (standard antibiotics) with tested extract and the mean activity of methanol extract varies in an inverse manner (Supplementary material Table S4). As evident from the Supplementary material Table  S2, methanol extract of tested plant had significant content of phenolics (258.5 mg GAE/g of dry extract), flavonoids (306.26 mg QE/g of dry extract) and flavonols (144.21, mg QE/g of dry extract). As evident from the literature, some reports suggested that polyphenolic compounds detected in the extract have been documented to possess antibacterial activity (Okwu 2004;Afolabi et al. 2007). The toxicity of polyphenols in microorganisms is generally due to iron deprivation or hydrogen bonding with microbial enzymes affecting their activity or enzyme inhibition by the oxidation of phenolic compounds (Scalbert 1991;Davidson and Naidu 2000). According to Cowan (1999) the antibacterial activity of flavonoids is due to their ability to complex with extra cellular and soluble proteins as well as with the bacterial cell wall.Therefore the conspicuous antimicrobial activity exhibited by methanol extract in the present study may be attributed to the presence of polyphenolic compounds.

Conclusions
This report shows the phytochemical composition and biological activities of methanol extract of F. micrantha, 55 constituents have been identified in this study. Plant extract showed remarkable antibacterial activity against plant pathogenic bacteria. It is rich in polyphenolic compounds with different molecular structures and react in different ways when reducing Fe þ3 , H 2 O 2 and DPPH radicals. Consequently, natural extract obtained from F. micrantha is excellent antioxidant agents.This is the first report about the phytochemical analysis and biological effects of Himalayan F. micrantha extract. The results of this study suggest the possibility of using the plant extract or some of its components for the applications of medical and agriculture field as an antimicrobial and antioxidant ingredients.