Bioactive saponin profiling of endophytic fungi from Asparagus racemosus

Abstract Thirty-five distinct endophytic fungi were isolated from the roots of Asparagus racemosus. Five out of 35 isolates were found to be efficient saponins producers and they were identified as Aspergillus terreus (E.F-1), Aspergillus flavus (E.F-7), Penicillium sp. (E.F-12), Talaromyces pinophilus(S-26), and Aspergillus terreus (Y-2) based on 18 sr RNA sequencing. The crude extracts of endophytic fungi were screened using High-performance liquid chromatography (HPLC) for quantitative analysis of saponin. The crude extracts of endophytic fungi were also characterised using FT-IR spectroscopy and mass spectrometry. The IR spectra of all five endophytic fungi crude extracts revealed the presence of –OH,-CH Alkyl,-CH3,-C-O-C,-C=C,-C=O stretching, which indicated the presence of saponin. Eight types of saponins recognised by mass spectrometry were Cyclamine saponin, Aspoligonin A, Sarsapogenin, Asparacosin A, Schidigera saponinD5, Aspargoside A, Dioscin, and Protodioscin. Endophytic fungi extracts also exhibited antimicrobial activity and antioxidant activity. Graphical Abstract


Introduction
Asparagus racemosus Wild is a diploid spinous under-shrub distributed throughout India's tropical and subtropical parts and is commonly called Shatavari.This plant is recommended for cure in threatened abortion, lactation in women, and normalising the uterus and hormone changes that occur during pregnancy (Garg and Gupta 2010).The roots of this herb are reported to have several biological activities like antiulcer, anticancer, antioxidant (Parihar andHemnani 2004), anti-diabetic, antitussive, antihyperlipidemic, antistress, antidepressant, antianxiety, immunomodulatory, anti-inflammatory, anti urolithiatic, antibacterial, andantidiarrhea (Mandal et al. 2000).Major constituents of Asparagus racemosus include lignin, acemannan, oligosaccharides, quercetin, rutin, racemoside, diosgenin, hyperoside, sarsasapogenin, steroidal saponins, and triterpene saponins (Iqbal et al. 2017).Asparagus racemosus is now considered endangered in its natural habitat and recognised as vulnerable (Bopana and and Saxena 2008).Therefore, there is a need to discover a strategy to decrease the dependency on plants.Research on endophytic fungi is nowadays focused to overcome this problem.It is believed that screening for secondary metabolites from endophytic fungi from medicinal plants is a promising way to overcome the dependency on the plants.Secondary metabolites producing endophytic fungi are an excellent approach to fulfill the excessive demand for medicinal plants.Many researchers have proved that endophyte fungi are a new and potential source of novel natural products (Guo et al. 2008;Suryanarayanan et al. 2009).Secondary metabolites isolated from endophytes belong to diverse structural classes, including alkaloids, peptides, steroids, terpenoids, phenols, quinones, saponins, and flavonoids (Gunasekaran et al. 2017).Some endophytic fungi have developed the ability to produce similar bioactive secondary metabolites as those from the host plants (Ludwig-M€ ulle 2015).Keeping this in view, the present investigation aimed to isolate, screen, characterisation, and evaluation of the microbial activity of saponins-producing endophytic fungi from the roots of Asparagus racemosus.

Isolation and screening of endophytic fungi
A total number of 35 endophytic fungi were isolated from the roots of Asparagus racemosus.After screening crude extracts of the isolates by HPLC (High-Performance liquid chromatography), only five were found to be efficient saponin producers.Table 1 depicts saponin production in 1 mg of crude extract of each isolate.A Maximum amount of saponin (5441.54ng/mg) was observed in isolate E.F-1 (Aspergillus terreus).The retention time was found to be 1.7 ± 0.1 min.Figures S1-S7 showed the calibration curve and chromatogram of all five isolates (extracts) along with the standard.Wu et al. (2012) reported saponin-producing endophytic fungi G22 (Penicillium sp.) from Aralia elata.They observed that G22 produced the highest concentration of saponins (2.049 mg/mL).Complete details of the five isolates given in Tables S1 and S2.

Molecular phylogenetic analysis of endophytic fungi
The interpretation of the evolutionary tree of E.F-

Mass spectrometry analysis
By comparing the obtained molecular mass from mass spectrometry of crude extracts of endophytic fungi with available data, we confirmed the presence of saponins compounds.We have found eight types of saponin compounds in all five endophytic fungi extracts (Table S5).The mass spectrum of eight saponins compounds was shown

Conclusions
The present study supports the hypothesis that endophytes of ethno-healthful plants may be a good source of medicinal valuable useful secondary metabolites.Asparagus racemosus's saponins were also observed in endophytic fungi.This may be due to long co-evolution (Strobel and Daisy 2003) and gene transfer mechanisms in endophytic fungi from their host plant (Kusari et al. 2009).This is the first study to isolate similar saponins producing endophytic fungi from the roots of Asparagus racemosus.
Recently researchers extracted several secondary metabolites of plants from endophytic fungus and these metabolites are widely used for the treatment of many severe diseases (Aly et al. 2011).

Geolocation information
Bio & Nano Technology Department, Guru Jambheshwar University of Science and Technology, Hisar, Haryana, India.

Figure
FigureS14depicts the IR spectra of all five endophytic fungi extracts.TableS4shows the bands with their ranges.The IR spectra of each endophytic fungus extract compared with the IR spectra of saponin standard revealed similar functional groups OH, C-H, CH 3 , C¼C, C¼O, C-O, and C-O-C indicating the presence of saponins.The extra peaks seen in E.F-1 and E.F-7 indicate the purification requirement of crude extract.C-O-C reveals glycoside linkages.Our results are in agreement with the previous reports on saponins.They observed -OH, -C¼O, C-H, C¼C, and C-O-C absorption, which verified the presence of saponin in medicinal plants(Kareru et al. 2008).OH, C¼O, C-H, C¼C, and C-O were also observed in Sapindus mukorossi indicating the presence of saponins(Almutairi and Ali 2015).Bajad and Pardeshi (2016) also reported -OH, -CH 3, C¼O, C¼C, and C-O stretching in the crude extract of Sapindus emarginatus.The existence of O-H stretching, C¼C stretching, CH 2 , CH 3 , C-O stretching, ¼CH bending represented the occurrence of steroidal saponin (PSI, PSII, PSVI, and PSVII) in Paris plant(Yang et al. 2018).

Table 1 .
Concentration of saponin in all endophytic fungi extracts.