Identification of the Diterpenoids Produced by Endophytic Fungus of Torreya fargesii and I

Identification of the Diterpenoids Produced by Endophytic Fungus of Torreya fargesii and Its Inhibitory Effect on Hela CellsWeihua JIN ，Junwei CAO *，Yi TU ，Yi TANG ，Ｒan GE ，Jingwen CHEN ，Fu XU ，Qijie XIONGDepartment of Life Science and Chemistry ，Wuhan Donghu University ，Wuhan 430212，ChinaAbstract ［Objectives ］The purpose of this study is to dissociate endophytic fungus producing diterpenoids from Torreya fargesii tissue and examines its inhibiting effect on tumor cells．［Methods ］Plant endophytes were isolated and purified to study their resistance to Gram-positive （G +）and Gram-negative bacteria （G －）．High performance liquid chromatography （HPLC ）was used for analysis of the retention time ，rela-tive peak area and percentage content of its metabolite．By liquid chromatography-mass spectrometry （HPLC-MS ），the material characteristic of the ion pair information of the metabolites was measured．The bacterial strain was also classified．［Ｒesults ］The results showed that the sec-ondary metabolites produced by the strain BP6T3possessed double resistance to G +and G －bacteria．The strain was identified as Penicilliumsp by preliminary classification．Through HPLC analysis ，the retention time of fermentation extracts was 12．8min with almost the same as the standard of taxol．According to the chromatograph ，the relative peak area was 12887．11，the average relative percentage was about 15．8%，and the content of taxol analogs in fermentation broth reached 16．59mg /L．The material characteristic of the formation of ion fragments of tax-ane analogues in metabolic extracts was identical to that of the taxol standard determined by HPLC-MS．It can be initially determined that strainBP6T3can produce taxane compounds．Taxol substance produced by this strain had obvious inhibitory effect on Hela cells with the concentra-tion increasing．Different precursors had a significant effect on the production of paclitaxel metabolites in this strain．L-phenylalanine was usedas the precursor and the yield increased most ，with an increase rate of 425．7%．［Conclusions ］The strain is expected to be used for mass production in antitumor drug taxol．Key words Endophytic fungus ，Penicillium sp ，Taxol ，Hela cells ，Inhibitory effectＲeceived ：January 15，2019Accepted ：June 10，2019Supported by Provincial College Students ’Innovation and Entrepreneurship Training Program of Colleges and Universities in Hubei Province in 2017（2013）；"Strategic Emerging （Pillar ）Industrial Talent Training Program"of Colleges and Universities in Hubei Province ［Hubei Provincial Department of Education EJiaoGao （201711798030）No．11］；Pilot Funded Project of "Comprehensive Professional Ｒeform"of Provincial Department of Education and Provincial Department of Finance ［EJiaoGaoBan （2014）No．6］；Hubei Educational Science "Twelfth Five-Year Plan"Project （2014B272）；SchoolYouth Natural Science Foundation （2013dhzk003）．*Corresponding author．E-mail ：215351231@qq．com1IntroductionTaxol was first isolated from Taxus ，but the content of taxol in Taxus is very low ，only 0．003－0．069%（V /V ）［1－4］．The guar-antee of taxol raw materials has become a key factor in the success of the drug to the market．Isolation of endophytic fungus producing taxol or analogues is a new way to effectively solve the problem oftaxol resources in recent years ［5］．The hosts of taxol-producing fungi are mostly found in the genera Taxus and Austrotaxus ［4－7］ofthe gymnosperms family Taxaceae．At present ，the endophytic fungus isolated are mainly from these genera ，but the content of taxol or analogues is generally low ，which does not meet the re-quirements of industrial production ［8］．A large number of studies have shown that the addition of precursors has a certain effect on increasing the content of endophytic taxol．Zamir et al．［9］reported that the tetracyclic diterpene skeleton of taxol came from hydroxy-methylvaleric acid ，the acyl group at position C-10came from ace-tic acid ，and the acyl side chain at position C-13came from phen-ylalanine．Therefore ，when endophytic fungus were cultured ，adding these precursors to the medium could effectively increase the content of taxol ［15］．In this study ，endophytic fungi were isolated from the tissues of Torreya fargesii collected from Shennongjia National Geological Forest Park．Then the strains producing taxol or analogues were screened by high performance liquid chromatography （HPLC ），liquid chromatography-mass spectrometry （LC-MS ）and antitumoractivity analysis．In order to increase the yield of taxol or ana-logues ，precursor substances and other methods were used to in-crease the yield of taxol or analogues．2Materials and methods2．1Materials The plant tissues of Torreya fargesii ，such as roots ，stems ，leaves and bark ，were collected from Shennongjia National Geological Forest Park ，Hubei Province （providing plant identification experts ）．taxol control （Beijing Solarbio Technology Co．，Ltd．，lot No．170922），methanol of chromatographicpurity ，other reagents of analytical purity．potato dextrose agar （PDA ）medium （g /L ）：potato ，200；dextrose ，20；KH 2PO 4，1．0；MgSO 4·7H 2O ，0．5；agar ，15－20；natural pH．Without agar ，it acted as a PDA seed liquid medium．Czapek medium （g /L ）：NaNO 3，2．0；K 2HPO 4，1．0；KCl ，0．5；MgSO 4·7H 2O ，0．5g ；FeSO 4·7H 2O ，0．01；sucrose ，30；agar ，15－20；natural pH．LBmedium （g /L ）：yeast powder ，5；NaCl ，10；peptone ，10；pH 7．4．15－20g of agar powder was added to the solid．Liquid chromatograph （HPLC ）model is Nexera LC-30AUPLC ；mass spectrometer （HPLC-MS ）model is AB scie Tri-ple Qaud 4500（triple quadrupole mass spectrometer ）．Medicinal Plant 2019，10（3）：45－48DOI ：10．19600/j．cnki．issn2152－3924．2019．03．0112．2Methods2．2．1Pretreatment of Torreya fargesii materials．The roots，stems，leaves and bark of Torreya fargesii were washed with asep-tic water and dried with aseptic absorbent paper．And they were sterilized with75%alcohol for5min and2%hypochloric acid for 8min，then rinsed with aseptic water for3times．The collected roots，stems，leaves and bark were then cut into small segments of roughly the same size with an aseptic knife．2．2．2Isolation of endophytic fungi．The plant materials treated by the above methods were placed in PDA solid medium plate and cultured at28ħuntil mycelium grew．2．2．3Purification of endophytic fungi．When the fine mycelium grew at the edge of the section of the inoculum in the plate，the mycelium was selected by inoculation needle and inserted into the newly prepared PDA solid plate．Then it was purified several times on PDA inclined medium until the pure culture was obtained．2．2．4Identification of endophytic fungus．The characteristics of endophytic fungus in plate culture were observed，and Shen Ping’s method［10］was used to observe the morphology of mycelium and conidia under microscope．2．2．5Extraction of taxol analogues from endophytic fungus．The purified endophytic fungi were activated in PDA liquid medium and inoculated in PDA liquid medium at28ħfor oscillating cul-ture．The filtrate was filtered and collected，and the filtrate was extracted with ethyl acetate to collect the upper organic phase．The organic solvent was evaporated in a rotary evaporator．2．2．6Detection by high performance liquid chromatography （HPLC）．（i）Chromatographic conditions were as follows：chro-matographic column，ODS（C18）column4．6mmˑ250mm，5μm；column temperature，room temperature；mobile phase，methanol/water=65/35（V/V）；flow rate，1．0mL/min；sample injection volume，20μL；UV detection wavelength，227nm．（ii）Sample treatment：the ethyl acetate extract was dissolved in the mobile phase，the supernatant was absorbed by centrifugation at 13200r/min for2min，and filtered by0．22μm pore size filtera-ble membrane．（iii）Quantitative analysis：the peak area was cal-culated according to the chromatogram．2．2．7Determination of fat-soluble metabolites by high perform-ance liquid chromatography-mass spectrometry（HPLC-MS）．（i）Chromatographic conditions were as follows：chromatographic col-umn，ODS（C18）column2．1mmˑ50mm，1．7μm；column temperature，room temperature，mobile phase，acetonitrile/water =65/35（V/V）；flow rate：0．2mL/min；sample injection vol-ume，1μL．（ii）Sample and mobile phase treatment：the extrac-ted product of ethyl acetate was dissolved in the mobile phase，the supernatant was absorbed by13200r/min centrifugation for 2min，and filtered by0．22μm pore size filterable membrane．（iii）MS parameters：（a）MS ion source：curtain gas（CUＲ），30．00；collision gas（CAD）：8．00；electrospray voltage（IS），5500．00；ion source temperature（TEM），550．00；nebulizer （GS1），40．00；auxiliary gas（GS2），45．00；inlet voltage （EP），10．00；collider outlet voltage（CXP），13．00．（b）Scan mode MＲM．2．2．8Inhibitory effect of extract on Hela tumor cells．Hela cells in logarithmic growth phase were inoculated in96-well plate at100μL/well and cultured in5%CO2incubator at37ħfor24h．Then different concentrations of taxol analogues（10μL）wereplaced into the sample wells．After being cultured in5%CO2in-cubator at37ħfor24h，the culture plate was taken out，the su-pernatant was absorbed，and90μL of fresh culture medium and 10μL of CCK-8were added to each well．After4h of culture un-der the same conditions，the A450value was read at450nm by mi-croplate reader（Elisa），and the cell growth inhibition rate was calculated．Cell growth inhibition rate was calculated as follows：Hela cell inhibition rate（IＲ）=（Control well A value－Sample well A value）/Control well A valueˑ100%．2．2．9Effects of precursors on the production of taxol metabolites by endophytic fungus．The endophytic fungi were inoculated in PDA liquid medium at28ħfor180r/min and the endophytic fungi were cultured for5d in oscillatory culture．Different precur-sors were added to the fermentation medium and sucrose solution was added at the same time to continue the culture．At the end of culture，taxol analogues of endophytic fungus were extracted．High performance liquid chromatography（HPLC）was used to de-termine taxol analogues in metabolic extracts and the content of taxol analogues was determined by HPLC．3Ｒesults3．1Isolation and purification of endophytic fungus from Torreya fargesii The material of Torreya fargesii was properly treated．After7d of culture，18strains of endophytic fungus were isolated from the tissues of Torreya fargesii．7plants were isolated from leaves，9from stems and2from bark．At the same time，no colony grew in the control group（uncut tissue），which confirmed that the isolated strains were endophytic fungus．3．2Identification of endophytic fungus BP6T3．The active BP6T3in18strains of endophytic fungus was classified and identi-fied．After observation of the characteristics of plate culture，it was found that the colony color of the strain was green and the col-ony texture was velvety（Fig．1）．Under the microscope，the hyphae were multicellular branches，the hyphae had transverse septum，and conidial pedicels also had transverse septum．There were no podocytes at the base and no enlarged apical sac at the tip，and its conidial pedicel was branched many times，resulting in two rounds of asymmetric pedicels，shaped like brushes．Conidia were spherical or oval，smooth or rough，and most of them were blue-green when they grew．The hyphae had erect multicellu-lar conidium pedicels on which each had a string of gray-green conidia（Fig．2），which could be preliminarily identified as Peni-cillium sp［11］．64Medicinal Plant2019Fig．1Bacterial lawn of strainBP6T3Fig．2Microscopic picture of strain BP6T33．3Analysis of taxol analogues in endophytic fungus BP6T3After the strain BP6T3was activated in PDA liquid medium ，the strain was cultured in PDA liquid medium at 28ħand 180r /minfor 7－10d．The filtrate was filtered and collected ，one third vol-ume ethyl acetate was added for countercurrent extraction ，the up-per organic phase was collected ，and the organic solvent was re-moved at 35ħin the rotary evaporator．The fermentation crude extract of strain BP6T3was detected by HPLC chromatography and determined under the same condi-tions of taxol control．The retention time （Ｒt ）of taxol control was 11－14min （Fig．3），and the Ｒt of strain BP6T3was 12．8min （Fig．4）．Therefore ，it was preliminarily determined that the me-tabolites of strain BP6T3contained taxol substances．The relativecontent of metabolites was 15．8%，and the content of taxol ana-logues in fermentation broth was 16．59mg /L （Fig．4）．Fig．3HPLC chromatogram of taxol referencesubstanceFig．4HPLC chromatogram of metabolic extract of strain BP6T33．4Determination of the characteristics of ion fragments formed by taxol analogues by HPLC-MS The characteristicsof taxol analogues forming ion fragments in the metabolic extractsof Penicillium BP6T3were determined by HPLC-MS ，and the re-sults showed that the ion fragment information of taxol analogues was completely consistent with that of taxol standard substance （Table 1）．The taxol analogues produced by Penicillium BP6T3can be preliminarily characterized as taxol．Table 1The ion pair information of the taxol standard and taxol analogues produced by strain BP6T3by the mass spectrometric analysisMass-to-charge ratio of parent ion ∥m /z Mass-to-charge ratio of daughter ion ∥m /zScanning time ∥msec Declustering potential （DP ）Collision energy （CE ）876．3307．95015036876．3591．25015036876．3531．250150403．5Inhibitory effect of extract on Hela tumor cells Helacell is a cell line isolated from human cervical cancer tissue ，which can be subcultured continuously ，the cell line will not die of ag-ing ，and can divide indefinitely．Compared with other cancer cells ，it has rapid proliferation and other characteristics．It has been widely used in tumor research ，biological experiments or cell culture ，and has become a very important tool in medical research．The inhibitory effect of taxol analogues on Hela cells was also used as the basis for judging the antitumor effect of taxol analogues．The inhibitory effect of strain BP6T3fermentation broth ex-tract on Hela tumor cells was tested by CCK-8reagent method ，and the inhibition rate on cell growth was calculated （Table 2）．Table 2Inhibitory effect of taxane from strain BP6T3on Hela cellsConcentration of sample ∥μg /mL OD 450value Ｒate of inhibition ∥%CK 1．100－0．10570．78324．141．0570．66735．4710．570．44756．6874Weihua JIN et al．Identification of the Diterpenoids Produced by Endophytic Fungus of Torreya fargesii and Its Inhibitory Effect on Hela CellsThe OD value calculation showed that taxol or taxol analogues produced by the strain had obvious inhibitory effect on Hela cells，and the inhibition rate increased with the increase of concentration．3．6Effects of precursors on the production of taxol metabo-lites by endophytic fungi Three precursor solutions，sodium benzoate，sodium acetate and L-phenylalanine，were prepared．Penicillium BP6T3was inoculated in PDA liquid medium at28ħfor180r/min．On the5th day of fermentation，the three precur-sors were added to the fermentation medium．The final concentra-tions of L-phenylalanine solution，sodium acetate solution and so-dium benzoate solution were3．0mg/L，3．5g/L and32．0mg/L，respectively．Sucrose solution was added to each fermentation me-dium，and the final concentration was5．0g/L．On the10th day，the fat-soluble metabolites of Penicillium BP6T3were extracted．High performance liquid chromatography （HPLC）was used to detect taxol in metabolic extracts qualitative-ly and quantitatively，and the growth rate of taxol was calculated （Table3）．It can be seen that with L-phenylalanine as precursor，the yield of taxol increased most，with a growth rate of425．17%．Table3Effects of different precursors on taxol produced by strain BP6T3Precursor materials Peak area Growth rate∥%CK12887．11－L-phenylalanine67678．78425．17Sodium acetate35735．06177．29Sodium benzoate35774．63177．604DiscussionsIn this study，endophytic fungi were isolated from the tissues of Torreya fargesii．Endophytic fungi with the ability to produce taxol were isolated from plants other than Taxus，which greatly expanded the range of resources to produce taxol or analogues．This is con-ducive to the wider use of microbial resources for the production of antitumor drugs［18－20］．Some studies have shown that the content of taxol in fermentation broth can reach more than9mg/L，and the cost recovery can be guaranteed in production［9－10］．At present，the main reason why endophytic fungus cannot be used in produc-tion is still the too low yield，generally only a few hundred micro-grams per liter［14－15］．The strain BP6T3obtained in this study could produce taxol analogues very stably．The content of taxol an-alogues in the fermentation broth was as high as16．59mg/L．This was higher than the previously reported taxol production from endo-phytic fungus［9－14］．The taxol produced had obvious inhibitory effect on Hela cells and increased with the increase of concentra-tion．Moreover，different precursors had obvious effects on the pro-duction of taxol metabolites by this strain，and the yield increased most when L-phenylalanine was used as the precursor．Therefore，the fermentation of taxol or analogues by the plant endophyte Peni-cillium BP6T3obtained in this study can be carried out in a short time and on a large scale without the limitation of resources，envi-ronment，conditions，equipment and so on．Ｒeferences［1］ＲEN YY，WANG P，ZAN N，et al．A survey of studies on activities and toxicities of diterpenoid alkaloids［J］．Chinese Journal of Experimental Traditional Medical Formulae，2010，16（11）：210－213．［2］MA XC，WU LJ，JIA JM．Development in the research on toxoids［J］．Journal of Shenyang Pharmaceutical University，2002，19（2）：147－156．［3］STIEＲLE A，STIEＲLE D，STＲOBEL G，et al．Endophytic fungi of Pa-cific yew（Taxus brevifolia），as a source of taxol，taxanes，and other pharmacophores［M］．ACS Symposium Series，1994，25（42）：64－77．［4］GUO BH，WANG YC，ZHOU XW，et al．An endophytic taxol-producing fungus BT2isolated from Taxus chinensis var．mairei［J］．African Journal of Biotechnology，2006，5（10）：875－877．［5］MA TY，DONG ZL．The study of isolating endophytic fungus synthesizing taxol from plant［J］．Journal of Northwest University（Natural Science Edition），1999，29（1）：47－49．［6］ZHOU DP，SUN JQ，YU HY，et al．Nodulisporium，a genus new to China［J］．Mycosystema，2001，20（2）：277－278．［7］ZHOU DP，PING WX，SUN JQ，et al．Isolation of taxol producing fungi ［J］．Journal of Microbiology，2001，21（1）：18－19．［8］QIAO W，LING F，YU L，et al．Enhancing taxol production in a novel endophytic fungus，Aspergillus aculeatinus Tax-6，isolated from Taxus chinensis var．mairei［J］．Fungal Biology，2017，121（12）：1037－1044．［9］ZAMIＲLO，NEDEA ME，GAＲALAU FX．Biosynthetic building blocks of Taxus cannadensis taxanes［J］．Tetrahedron Letters，1992，33（36）：5235－5237．［10］SHEN P，CHEN XD，et al．Experiment of microbiology（version5）［M］．Beijing：Higher Education Press，2007：241－242．［11］KONG HZ．Chinese fungi Penicillium and related sexual type（35）［M］．Beijing：Science Press，2007：5－10．［12］KASAEI A，MOBINIDEHKOＲDI M，MAHJOUBI F，et al．Isolation of taxol-producing endophytic fungi from Iranian yew through novel molecu-lar approach and their effects on human breast cancer cell line［J］．Cur-rent Microbiology，2017，74（6）：702－709．［13］JAYANTHI G．Isolation and characterization of anticancer compound，taxol from an endophytic fungus Phomopsis longicolla［J］．FEBS Letters，2017，316（2）：147－151．［14］PATEL VK，SAＲIM KM，PATEL AK，et al．Chapter14Synthetic mi-crobial ecology and nanotechnology for the production of taxol and its pre-cursors：A step towards sustainable production of cancer therapeutics ［J］．Design of Nanostructures for Theranostics Applications，2018：563－587．84Medicinal Plant2019。