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Study on Endophytic fungi producing Orange Pigment isolated from Ginkgo Biloba L1 Jia-Jia Liu1, Shu-Juan Chen1, Han-Xiang Gong2 1Department of Pharmacy Engineering, college of chemistry and chemical engineering, central south university , Changsha (410083) 2 Suzhou Changshu bureau of Quality and Technique Supervision, Changshu (215500) E-mail: [email protected] Abstract More than seventy endophytic fungi were isolated by aseptic techniques from the phloem of the of Ginkgo biloba L.. They were cultured in the potato dextrose (PD) liquid medium and one endophytic fungus (Gh01) was proved can produce orange pigment. The orange pigment was identified as by chemical reaction and HPLC. This is the first report on quercetin glycoside produced by endophytic fungus. The effects of the carbon and nitrogen sources, metal ions, initial pH and incubation temperature on pigment production of the endophytic fungus were evaluated. The optimal temperature and initial pH for pigment production in PD liquid medium were 28℃ and 7.0, respectively. Through the orthogonal trial, 20 g/L Glucose and 5 g/L peptone were the most suitable carbon and nitrogen source, 1 g/L chloridize zinc could increase the yield of pigment. Under the optimal conditions established, the maximum yield of the pigment was 27.515 g/L after 120 hours’ successive culture. Keywords: Ginkgo biloba L; Endophytic fungi; Pigment; Quercetin glycoside; Optimization 1. Introduction endophytic fungi are eukaryotic organisms that live inside plant tissues and plant behaves as hosts[1]. The association is symbiotic and both organisms profit from the relationship. That is, the plant is thought to provide nutrient to the microorganisms, while the microorganisms may produce factors that protect the host plant from attack by animals, insects or other microorganisms[2]. Endophytes are presumably ubiquitous in , which populations depend on host species and location, and they have been recognized as a valuable source of novel bioactive metabolites[3]. Utilizing microorganisms (especially the fungi) to produce some chemicals and bioactive production by fermentation is promising because it could be further benefited by optimizing the fermentation techniques or meliorating the seed of microorganisms. Ginkgo is an ancient dioecious plant. At present, only Ginkgo biloba L. still exists as a living fossil plant. Botanists have studied it from different aspects in the world due to its unique characteristics[4]. Its fruits and seeds have been used for the treatment of asthma, cough and enuresis[5]. Since 1990s, the standardized extracts of Ginkgo. biloba L. leaves has becoming one of the most popular supplements for memory enhancement. The genistein, biochanin A, luteolin, quercetin, and are plant natural products with potentially useful pharmacological and nutraceutical activities. These natural products usually exist in plants as [6]. Quercetin glycoside is derived by enzymatic transglycosylation[7], and it is very important in medicines[8,9] and food industry[10]. The leaves of Chrysanthemum coronarium are shown to be rich in quercetin and its glycosides[11]. A series of Quercetin glycosides are isolated from the translucent of nobile as UV-absorbing substances [12]. Moreover, quercetin-type glycosides are isolated from the stems and leaves of Delphinium hybridum cv. “Belladonna Casablanca” (Ranunculaceae)[13]. Recently, a new lactoyl glycoside quercetin which is isolated from Melia azedarach leaves are reported[14]. However, there have been no reports on quercetin glycoside from endophytic fungi.

1 Corresponding Author.Tel.:+86-731-8836834; Fax: +86-731-8836834.

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In this study, an endophytic fungus which could produce orange pigment(Quercetin glycoside) is isolated, and its biological characters and culture conditions are discussed. The results will be useful for Gh01’s further using. 2. Materials and Methods 2.1. Plant material The of Ginkgo biloba L. trees (ø=50-80 cm) were collected in the Central South University, Hunan province, central China.

2.2. Isolation and identification of endophytic fungi The outer bark of each sample was excised by a sterilized sharp blade after washed with running tap water, sterilized with 75% (v/v) ethanol for 2 min and 2.5% sodium hypochlorous for 10 min, rinsed in steriled water for several times and cut into pieces (about 1cm×1cm). Then, each sample was incubated at 27℃ in the melted potato dextrose agar (PDA) medium supplemented with 200 µg/mL ampicillin and 200 µg/mL streptomycin to inhibit the bacterial growth until the mycelium or colony originating from the newly formed surface of the segments appeared. Several days latter, fungi were observed growing from the inner bark fragments in the plates. Individual hyphal tips of the various fungi were removed from the agar plates, placed on new PDA medium, and incubated at 27℃ for at least 10 d. Each fungal culture was checked for purity and transferred to another agar plate by the hyphal tips method which was described by Stroel G et al[15]. Fungal identification methods were based on the morphology of the fungal culture, the mechanism of spore production, and the characteristics of the spores. All experiments and observations were repeated at least twice. The purified endophytic fungi were numbered and stored in distilled water at 4℃ as agar plugs and 15%(V/V) glycerol at -80℃ as spores and mycelium.

2.3. Fermentation and treatment of the fermentation broth The inoculum was prepared by introducing the periphery of 7-day-old endophyte in Petri dish into 500 mL flasks, which contained 250 mL of potato dextrose liquid medium. After 4 days of incubation at 27℃ on rotary shaker at 120 rpm., 10 mL of incubated liquid (about 106 spores) as the seed was transferred into 250 ml flask which contained 150 mL potato dextrose liquid medium. After 7 days of growth at 27℃ , crude fermentation broth was filtrated thoroughly, distilled in decompressor at 40℃ , added absolute alcohol (v:v=1:1) and centrifuged at 3500 rpm for 10 min. The filtrate was collected and stored.

2.4. Screening of the aim strain During the fermentation, an endophytic fungus whose label was Gh01 producing pigment was screened out. The color of pigment was in red state at first, then turned to orange. This strain was isolated from the phloem of the root of Ginkgo biloba L., and it was the trial material in following researches.

2.5. Identification of the pigment Flasks(500 mL) containing 200 mL PD liquid medium were autoclaved twice at 121℃ for 20 min, then 10 mL of the seed liquid(about 106 spores) was added into per flask. After 7 days of growth at 27℃, crude fermentation broth was treated accordingly. Evaporation of the solvent in vacuo gave a brown residue (51.5 g), which was subjected to chromatography over silica gel

- 2 - http://www.paper.edu.cn column (500 g, 200-300 mesh) eluting with a chloroform-ethanol-water (13:6:1, 6 L) to collect the yellow and filemot fraction B (10.1 g). B was further separated over silica gel (100 g, 200-300 mesh) eluting with a methanol-water mixture (6:1, 2 L) to yield compound G-01 (948 mg). The identification of the pigment was carried out by chemical reaction and HPLC.

2.6. Optimizing of the culture medium In order to optimize the cultural conditions, the initial pH in the PDA medium was adjusted to (the desired value ) by addition of either 1 M HCl or 1 M NaOH. The flasks were incubated at various temperatures (20-32℃). 20 g/L Sucrose, maltose, lactose, glucose, rhamnose were studied as carbon sources. 5 g/L Beef extract, peptone, tryptone, ammonium chloride, ammonium nitrate, and yeast extract powder were used in this study to select the best nitrogen source. Metal ions (bluestone、zinc、bitter salt、chloridize calcium) were added into the culture medium to judge whether the existent of metal ions could influence the yield of pigment. During time factor study, the flasks were incubated at design temperature on a rotary shaker at 120 rpm for 7 days. After the appearance of pigment, the UV absorbability of the pigment was mensurated every 12 h. All experiments were performed at least in duplicate to ensure reproducibility. 3. Results and Discussions 3.1. The identification of Gh01 More than seventy endophytic fungi were isolated, and one endophytic fungus (Gh01) was screened out for producing orange pigment. The colony of the Gh01 was white in the initial period, and then turned to gray-green and became the shape of felt. Few protruding fetlocks were in its center while white flosses were on its edge. Few yellow liquid was secreted out on the surface of the colony. At the back of the substrate, bright red pigment could be seen in the earlier period, and its color became yellow-brown latter. The mycelium of hypha developed, with dissepiment, more branching, polynuclear, furthermore, taking on the fancy lotus root burl. The peduncle of conidiophore was about 13-25 µm in length, with diaphragm. Its top did not expand, having broom-like branchs, belonging to the structure of two rounds and asymmetric. The shape of the spores was rotundity or ellipse, bunchiness, lubricity, and the size was 2.0-2.5 µm. The pigment was produced extracellularly (Fig 1). According to those characters, Gh01 would belong to penillicium[16].

Fig. 1: Conidiophore and spore of Gh01

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3.2. The identification of the pigment UV measurement The absorbance wavelength of G-01(MeOH) was measured on the UVCRT756 UV spectrophotometer. The range of the scan wavelength was 190-500 nm. The result was shown in Fig 2. The peak absorption was at 252 nm、265 nm、351 nm, those was the characteristic absorbability wavelengths of the flavonol. Chemical qualitative reaction The G-01 (ethanol solution) was taken to react with some chemical reagents, including: HCl-Mg powder, NaOH, AlCl3 and FeCl3. The results of the chemical qualitative reaction (Table 1) showed that the pigment contained flavonol derivatives. TLC All comparative TLC analyses were carried out on GF254 silica gel plate developed in solvent system A(chloroform/methanol/acetic acid 7:2:1:1,v/v/v/v) and B(chloroform/methanol 60:40, v/v). The plates were detected under the UV lamp on 254 nm wavelength.

Table 1: The result of chemical qualitative reaction Test Deoxidizing reaction Hydroxybenzene complexing Color reaction methods reaction reaction

reagent HCl-Mg FeCl3 AlCl3 NaOH phenomena orange brown Kelly light brown fluorescence conclusion May had flavone May had flavone May had May had derivatives derivatives flavone or flavonol flavonol derivatives derivatives

Gh01 was first analyzed by TLC on GF254 plate with a solvent as the mobile phase. Quercetin and as the contrast samples. The dominating component of the pigment revealed Rf=0.14, with Kelly fluorescence, it’s Rf value was little smaller than rutin’s (Rf=0.16). In order to make the glycosides of the pigment clear, the hydrolyzed Gh01 was also analysed by TLC using the solvent system B. The results showed that there were glucose (Rf=0.58) and rhamnose (Rf=0.86) in the hydrolyzed Gh01 according to the reference[17]. HPLC For UV-photodiode array detection, HPLC analyses were performed on a HPLC-8A System controller combined with a SPD-6AV UV detector (351 nm). Separation were carried out using a Nova pac C18 (150mm×4mm) reversed-phase column. Elution was carried out at a flow-rate of 1.0 mL/min with the methanol/ water ( 60:40.) consisting of 0.2% phosphate acid. The standard samples (Quercetin) was bought from National Institute for the Control Of Pharmaceutical and Biological Products. The result of HPLC was showed in Fig 3. These results revealed that the endophytic fungus Gh01 could synthesize quercetin glycoside in vitro. And this is the first report on quercetin glycoside produced by endophytic fungi.

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Fig. 2: Chromatogram of UV of pigment

a b Fig. 3: The HPLC of Quercetin (a) and Gh01 (b)

3.3. Optimizing of the culture conditions The Gh01 was inoculated to PD liquid, Martin liquid, Czapek liquid, Yeast sucrose liquid and starch liquid media separately. The growth rate of the strain in those media was measured. The result showed that the strain could grow in each medium but growth rates were different. In the PD liquid, the strain grew fastest, and the mycelia were most abundant. So the PD liquid medium was the best culture medium for Gh01. Since yield was also affected by the culture conditions, including the temperature, time, initial pH, the carbon and nitrogen source, and metal ions. All of them were chosen for optimization. As shown in Fig 4, the optimum incubation temperature was 28℃ . At this temperature, the strain yield pigment after inoculation for about 96 h, and its production was highest compared with the other temperatures. The optimum incubation time was 120 h (Fig 5), when the yield run up to the maximum under the optimum temperature. The optimum initial pH was 7.0 (Fig 5). The optimum carbon and nitrogen source for the maximum pigment production by Gh01 were 20 g/L Glucose and 5 g/L peptone (data not shown).

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Fig. 4: The effect of temperature on the quercetin production

a b Fig. 5: The effect of culture time(a) and initial pH(b) on the quercetin production

The addition of metal ions could influence the yield of quercetin (Table 2): (1) Mg2+ had no influence on the growth of the strain or the yield of the pigment. (2) The pigment would never be 2+ 2+ produced while Cu was added to the broth. (3) The presence of Cu may inhibit some enzymes which were important to the production of the pigment. The addition of the Zn2+ would increase the yield and shorten the time for the appearance of pigment. So Zn2+ was benefit for the yield of quercetin.

Table 2: Effect of metal ions on the produce Metal ions Cu2+ Zn2+ Mg2+ Ca2+ none absorbency/AU - 0. 70 0.55 0.42 0.56

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3.4.Project of orthogonal trial. The orthogonal trial of L9 (33) was used to investigate the effects of three factors (carbon source, nitrogen source and metal ion) on yield of quercetin [Tables 3-6].

Table 3:Project of orthogonal trial Factors Levels 1 2 3 A glucose 1% 2% 3% B peptone 0.25% 0.5% 0.75 C Zn2+ 0.5×10-3g/ml 1×10-3g/ml 1.5×10-3g/ml

3 Table 4: Design of L9(3 ) table head and trial results No. Factors Results A B C absorbency/AU 1 1 1 1 0.26 2 1 2 2 0.57 3 1 3 3 0.23 4 2 1 2 0.48 5 2 2 3 0.67 6 2 3 1 0.37 7 3 1 3 0.22 8 3 2 1 0.36 9 3 3 2 0.32

Table 5: Analysis on results of orthogonal trail A B C K1 1.06 0.96 0.99 K2 1.52 1.60 1.37 K3 1.47 0. 92 1.12 R 0.46 0.68 0.38

Table 6: Analysis of variance Factors DEVSQ Degrees Of F Ratio F Critical Significance Freedom Points A 0.069 2 13.800 9.000 * B 0.097 2 19.400 9.000 * C 0.025 2 5.000 9.000 error 0.01 2

F0.10（2, 2）=9.000 As shown in Table 6, the sequence of the factors influenced on the production of quercetin was B (nitrogen source)> A (carbon source)> C (metal ions), and the mass percent of peptone and glucose were the Significance factors (f, a=0.10). In primary culture, the optimal formula of the three factors are 20 g/L glucose, 5 g/L peptone, 1 g/L ZnCl2. Under this condition maximum pigment production at 27.515 g/L was achieved after 120h’s successive inoculation.

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Quercetin has been exploited for its medicinal and nutritional activities[18,19]. It has a hydroxyl group at the 3-position of its Cring, and this may be important for its anticarcinogenic Activity[20]. Glycosylation of quercetin has been reported to increase its absorption in humans[21]. Moreover, quercetin-glycosides may affect the colon or other biological targets in a different manner when compared to quercetin aglycone[22]. Many studies have been aimed at isolating such new natural products from plants. But nature produces in plants are very complex and some produces’ content are very low, so direct isolation of products from plants will not answer our needs, but destroy our natural resources, endanger our entironment. Fortunately, studies on production of glycosides in E. coli which allow us to overcome these problems have recently been reported[5,23,24]. But using endophytic fungi to produce quercetin glycosides has never been described. This paper shows an endophytic fungus which could produce quercetin glycosides from Ginkgo Biloba L.. After the optimization of culture conditions its production reach 27.515 g/L. It should be another efficient approach to produce quercetin glycosides. And the industry-scale production of quercetin glycosides by endophytic fungi is expected. This study will be of benefit to the use of endophytic fungus Gh01. However, further research is needed on the isolation and structure identification of quercetin glycosides producing by endophytic fungus Gh01.

Acknowledgements This work was financed by a Grant for Key Research Items from the Ministry of Education (No.03126). References [1] Petrini O. (1996) Ecological and physiological aspects of host specificity in endophytic fungi. In: Redlin SC, Carris LM, eds. Endophytic Fungi in Grasses and Woody Plants: Systematics, Ecology, and Evolution. St. Paul, Minnesota: APS Press: pp.87–100 [2] Yang XS, Strobel G, Stierle A, Hess WM, Lee J, Clardy J (1994) A fungal endophyte-tree relationship: Phoma sp. In Taxus wallachiana. Plant Science, 102: pp. 1-9. [3] Bills GF, Polishook JD (1991) Microfungi from Carpinus caroliniana. Canadian Journal of Botany, 69: pp. 1477-1482 [4] Jin J, Jiang H, Yu SQ, Zhou GM (2008) Sex-linked photosynthetic physiologic research and the evolutionary ecological analysis in living fossil plant, Ginkgo biloba L.. Acta Ecologica Sinica, 28: pp. 1128-1136 [5] Zimmermann M, Colciaghi F, Cattabeni F, Di Luca M (2002) Ginkgo biloba extract: from molecular mechanisms to the treatment of Alzheimer’s disease. Cellular and Molecular Biology, 48: pp. 613-623 [6] He XZ, Li WS, Blount JW, Dixon RA (2008) Regioselective synthesis of plant (iso)flavone glycosides in Escherichia coli. Applied Microbiology and Biotechnology, 80: pp. 253–260 [7] Suzuki Y, Suzuki K (1991) Enzymatic formation of 4G-α-D-glucopyranosyl-rutin. Agricultural and Biological Chemistry, 55: pp. 181-187 [8] Shimoi K, Shen BR, Toyokuni S, Mochizuki R, Furugori M, Kinae N (1997) Protection by alpha G-rutin, a water-soluble antioxidant flavonoid, against renal damage in mice treated with ferric nitrilotriacetate. Japanese Journal of Cancer Research, 88: pp. 453-460 [9] Nagasawa T, Tabata N, Ito Y, Nishizawa N, Aiba Y, Kitts DD (2003) Inhibition of glycation reaction in tissue protein incubations by water soluble rutin derivative. Molecular and Cellular Biochemistry. 249: pp. 3-10 [10] Morita N, Nakata K, Hamauzu Z, Toyosawa I (1996) Effect of α-glucosyl rutin as improvers for wheat dough and breadmaking. Cereal Chemistry, 73: pp. 99-104 [11] Gins VK, Kolesnikov MP, Kononkov PF, Trishin ME, Gins MS (2000) Hydroxyanthraquinones and flavonoids of garland chrysanthemum. Applied Biochemistry and Microbiology, 36: pp. 296-305 [12] Iwashina T, Omori Y, Kitajima J, Akiyama S, Suzuki T, Ohba H (2004) Flavonoids in translucent bracts of the Himalayan Rheum nobile () as ultraviolet shields. Journal of Plant Research, 117: pp. 101-107 [13] Yoshimitsu H, Nishida M, Hashimoto F, Tanaka M, Sakata Y, Okawa M, Nohara T (2007) Chromone and flavonol glycosides from Delphinium hybridum cv. “Belladonna Casablanca”. Journal of Natural Medicines, 61: pp. 334-338 [14] Salib JY, Michael HN, El-Nogoumy SI (2008) New lactoyl glycoside quercetin from Melia azedarach leaves. Chemistry of Natural Compounds, 44: pp. 13-15 [15] Strobel G, Yang XS, Sears J, Kramer R, Sidhu RS, Hess WM (1996) Taxol from Pestalotiopsis microspora, an endophytic fungus of Taxus wallachiana. Microbiology, 142: pp. 435–440 [16] Wei JC (1982) Fungal Identification Manual. Shang Hai: Shanghai Scientific and Technological Press [17] Zhu J, Cao KM, Zhou RQ, Cai WC, Yuan HJ (1981) Biochemistry Experiment. Shang Hai: Shanghai

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