Isolation, characterization and antioxidant activities of the endophytic fungi from Hosta. Ventricosa
Nan Chen, Xiaoxia Liang*          
Natural Medicine Research Center, College of veterinary medicine, Sichuan Agricultural University, Chengdu611130, China
Abstract: A total of 13 types of endophytic fungi, classified into six genera, were isolated from Hosta. ventricosa based on 18S rDNA sequencing of ITS region and microscopic examination. The antioxidant activities of the crude extracts were investigated by T-AOC, DPPH and ABTS+ methods. The results showed that the crude extracts from Fusarium oxysporum (HoV1) and Epicoccum (HoV4), which presented higher phenol levels (1.90±0.03 mg/g, and 2.41±0.01 mg/g, respectively), exhibited stronger comprehensive antioxidant activities, with higher T-AOC values (63.46±0.26 U/g, and 66.05±0.71 U/g, respectively), lower EC50 values against ABTS+ (30.25±0.05 μg/mL, and 24.66±0.65 μg/mL, respectively)and DPPH radicals (83.42±0.24 μg/mL, and 59.136 μg/mL, respectively). Moreover, Fusarium oxysporum (HoV8) showed excellent scavenging activity against DPPH radicals (EC50 = 16.02 μg/mL), which was better than BHT. All of them could be useful in the developing of new natural antioxidant agents. 
Keywords: Hosta. Ventricosa; Endophytic fungi; Antioxidant activities; Total phenol contents  
CLC number: R284                Document code: A                 Article ID: 10031057(2018)1071108
1. Introduction
Endophytes alive in disease-free plants[1] are found almost in every plant and can produce the same or similar chemical compounds as their hosts, such as taxol[2], huperzine A[3], hypericin[4] and camptothecin[5]. Early study has reported that different types of natural compounds are found from endophytes, including flavonoids, alkaloids, terpenoids, steroids, fatty acids and so on, and most of them show strong biological activities, such as the anticancer compound vincristine[6], broad-spectrum antibacterial compound guanacastepene[7]and antioxidant compound isopestacin[8]. Therefore, plant endophytic fungi have been regarded as a novel resource for producing natural bioactive compounds[9].
Hosta. Ventricosa is a type of perennial herb of Hosta Liliaceae, which has been used as medical herbs in Inner
Mongolia for the treatment carbuncles, burns, sore throat and other diseases. Early phytochemistry research has identified its constituents as steroidal saponins, alkaloids, flavonoids and polysaccharides, and most of them show excellent biological activities, such as the inhibition of neurological diseases[10], anti-tobacco mosaic virus and inhibition of acetylcholin esterase[11].
However, few reports have mentioned the endophytic fungi from Hosta. Ventricosa until now. In order to figure out the medical use of its endophytic fungi, in our present study, we isolated endophytic fungi from Hosta. Ventricosa., and their in vitro antioxidant activities and the total phenolic content were investigated. 
2. Material and methods
2.1. Chemicals and materials
The plant was collected in the campus of Sichuan Agricultural University and authenticated by Associate Professor Qiaojia Fan from Sichuan Agricultural University. DPPH (purity greater than 97%) was purchased from BioDukly, and ABTS (purity greater than 98%) was obtained from Solarbio.
2.2. Isolation and identification of endophytic fungi
The fresh roots of Hosta ventricosa were cleaned under the tap water for more than 10 h. After the air-drying, the cleaned roots were surface sterilized by 5% sodium hypochlorite for 20 s, cleaned by sterile water for four times[12] and then cut into small pieces (0.5 cm×0.5 cm). The surface-sterilized samples were maintained on sterile potato dextrose agar (PDA) containing doxycycline hydrochloride (50 mg/L) and streptomycin sulfate (100 mg/L) at 28 ºC in the dark. The hyphal tip of endophytic fungus growing out from the plant tissue was cut by inoculation loop and transferred to a new PDA plate. The efficiency of surface sterilization procedure was examined for each sterilized plant segment following the imprint method, and the last washing water on the PDA was daubed as blank control.
All the isolated endophytic fungi (HoV1–HoV13) were stored in 20% glycerin solution at –80 ºC and kept at the College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan Province, China.
The total DNA of endophytic fungi (HoV1–HoV13) was isolated by OMEGA kit after the mycelium was ground in liquid nitrogen. DNA amplification was performed by PCR using PCR Mix (12.5 μL) and the primer pair ITS1 (1 μL): TCCGTAGGTGAACCTGCGG; and ITS 4 (1 μL): TCCTCCGCTTATTGATATGC, template DNA (1 μL) and molecular biology-grade water (9.5 μL).
Briefly, after an initial denaturation step 94 °C for 5 min, PCR amplification was carried out with 35 cycles at a melting temperature of 94 °C for 30 s, an annealingtemperature of 60 °C for 30 s, and an extension temperature of 72 °C for 1 min, followed by a ?nal extension at 72 °C for 10 min. Then the PCR products were sent to Tsingke Biotech (Chengdu, China) for sequencing.
To identify the types of endophytic fungi, the sequencing results were compared with the sequences in NCBI by Blast. Multiple sequence alignment using Clustal X2.0 was used in the present study. Phylogenetic tree was constructed using the neighbor joining (NJ) method by MEGA5.
2.3. Preparation of extract
Each endophytic fungal strain was inoculated in potato dextrose liquid medium at 25 ºC for 15 d under shakingat 170 r/min. The mycelia were collected by the filtration through four layers of gauze, then dried at 60 ºC and ground into a fine powder. The dried mycelia sample (1.0 g) was extracted with 75% ethanol (30 mL) for three times under ultrasonic extraction (30 min). The obtained extracts were filtered and concentrated to dryness under vacuum at 60 ºC and then stored at 4 ºC. The extraction rates were determined by A/1000 × 100%. A is the weight (mg) of the sample.
2.4. Determination of antioxidant activities
Antioxidant activities of the endophytic fungi were determined by total antioxidant capacity (T-AOC), the DPPH radical-scavenging activities and ABTS+ radical-scavenging activities. 
2.4.1. Total antioxidant activity
The total antioxidant activity was measured by T-AOC reagent kit (Nanjing Jiancheng), and the absorbance was measured at 520 nm using the T-AOC working solution containing no sample as blank. The results were measured as T-AOC = {[(OD1OD0)/0.01]/30} × (V/V1) × N, where OD1 is the absorbance of the samples, OD0 is the absorbance of the blank, V is reaction volume, V1 is sample volume, N is the sample dilution factor.
2.4.2. DPPH radical-scavenging activity
The DPPH radical-scavenging activity was measured according to the literature[13]. The 75% ethanol extract (1 mL, 23.44–750 μg/mL) was mixed with 0.004% ethanol solution of DPPH (1 mL). The mixture was reacted in the dark for 30 min, and the absorbance was determined at 517 nm. The butylated hydroytoluene (BHT) was used as a positive control.
2.4.3. ABTS+ radical-scavenging activity
The ABTS+ radical-scavenging activity was measured by Li’s method with minor modifications[14]. ABTS+solution (7.4 mM/L) was mixed with K2S2O4 solution (2.6 mM/L) in proportion of 1:1 (v/v) and kept in the dark at room temperature for 12–16 h. Each extract (0.2 mL) was mixed with ABTS+ (0.8 mL) in the dark for 10 min. The absorbance was measured at λ = 734 nm.
The ABTS+ radical-scavenging activity = [(A0 – A)/A0] × 100%.
A0 is the absorbance of the negative control, and A is the absorbance of the samples.
2.5. Determination of total phenolic contents
Taking gallic acid as standard, total phenolic contentsof the crude extracts from endophytic fungi were respectively measured by Folin-Ciocalteu’s colorimetric method[15] with minor modifications. The dried mycelia sample (1.0 g) was extracted by 75% ethanol like “2.3”, and then the final volume was filled up to 40 mL by adding 75% ethanol. The 75% ethanol extract (1 mL) was mixed with Folin-Ciocalteu’s reagent (0.1 M, 1 mL).After 1 min, 10% aqueous sodium carbonate (1 mL) was added, and then diluted to 25 mL with distilled water. After 30 min at room temperature, the absorbance was determined at 754 nm.
2.6. Data analysis
The data were analyzed by SPSS 10.0, and the tables were made by the Origin 8.0. Results were reported as mean±SD.
3. Results and discussion
3.1. Isolation and identification of fungal endophytes
A total of 13 types of endophytic fungi were isolated from the roots of Hosta ventricosa in this study. The ITS sequences were subjected to BLAST search in the NCBI GenBank database. Combining with morphological characteristics, the identification results of endophytic fungi were shown in Table 1. Figure 1 shows the phylogenetic relationships between these isolates and their related fungi. All the endophytic fungi were classified into six genera. There were seven types of Fusarium sp. (53.85%), one type of Ilyonectria sp. (7.69%), one type of Chaetomium sp. (7.69%), one type of Colletotrichum. (7.69%), one type of Epicoccum (7.69%) and two types of Rhizopycnis (15.38%). TheFusarium sp. was the dominant endophytic fungi from the roots of Hosta ventricosa 
Figure 1. Phylogenetic tree showing the relationship between endophytic fungi of Hosta ventricosa and their related fungal species.
Table 1. The identity of endophytic fungi and ethanol extraction rate of mycelium.
3.2. Determination of antioxidant activity
Taking BHT as standard, the antioxidant capacity of the ethanol extracts of the 13 fungal endophytes was assessed by their antioxidant capacity (T-AOC) and the scavenging abilities against DPPH and ABTS radicals.
3.2.1. Total antioxidant activity
T-AOC is a key indicator to value the enzymatic and non-enzymatic antioxidant substances. The decrease of T-AOC value marks the damage of antioxidant system, which may lead to cardiovascular diseases[16]. Table 2 lists the antioxidant capacity (T-AOC) of the ethanol extracts of 13 endophytes. The T-AOC values were within the range from 11.80±0.68 U/g to 92.61±0.51 U/g, among which, the value of HoV2 was significantly higher than that of others (92.61±0.51 U/g), but lower than that of BHT (349±0.71 U/g), followedby HoV4 (66.05±0.71 U/g) and HoV1 (63.46±0.26 U/g). Overall, the crude extract of Chaetomium sp. presented higher total antioxidant activities compared with Fusarium sp. Therefore, they might be useful in medical industry. 
Table 2. Overview of antioxidant and total phenolic content of ethanol extracts.
3.2.2. DPPH radical-scavenging activity
DPPH is a stable free radical, which has an unpaired valence electron at one atom of nitrogen bridge. DPPH free radical-scavenging method offers the first approachfor evaluating the antioxidant potential of a compound, an extract or other biological sources. Figure 2 shows the different scavenging activities of DPPH. All crude extracts except for HoV7, exhibited dose-dependent scavenging activities. Among them, the ethanol extract of HoV8 exhibited excellent radical-scavenging activity (IC50 = 16.02 μg/mL), which was even better than that of BHT (IC50 = 28.68 μg/mL) at the concentration of 375 μg/mL. Its scavenging activity could reach 99.31% at the concentration of 750 μg/mL. Such findings indicatedthat the extracts of HoV8 could be a promising resource of natural antioxidants, better than BHT.
Besides HoV8, the ethanol extracts of HoV1, HoV4, HoV11 and HoV13 showed better scavenging activitiesthan others, with IC50 valuesof 83.42 μg/mL, 59.13 μg/mL, 69.11 μg/mL and 74.16 μg/mL, respectively. Most of them, including HoV1, HoV8, HoV11 and HoV13, belong to Fusarium sp. Early reports have referred that the ethyl acetate extract from Fusarium sp can prolong the life of fruit fly by improving their erythrocyte life-span[17,18]. Our study may point out their antioxidant properties against DPPH in the near future.
Figure 2. DPPH radical-scavenging activity of the ethanol extracts of endophytic fungi, and the positive control BHT. 
3.2.3. ABTS+ radical-scavenging activity
ABTS + radical-scavenging activity is suitable for the analysis of hydrophilic and lipophilic samples, but short at the determination of free radicals in physiologicalconditions[19]. It was obvious that all 13 ethanol extracts of the endophytes showed different dose-dependent radical-scavenging activities against ABTS+ (Fig. 3). The best was observed from HoV4 (IC50 = 24.66 μg/mL), but lower than that of BHT (IC50 = 8.86 μg/mL). Besides HoV4, the ethanol extracts of HoV1, HoV5, HoV9 and HoV10 showed better scavenging activities than others, with an IC50 value of 30.25 μg/mL, 31.03 μg/mL, 33.09 μg/mL and 36.31 μg/mL, respectively. HoV1, HoV5 and HoV10 belong to Fusarium sp, and HoV4 belongs to Epicoccum. Both of those two types could be useful as ABTS+ radical-scavenging agents.
In summary, the crude extracts from Fusarium sp. (HoV1) and Epicoccum (HoV4) exhibited stronger comprehensive antioxidant activities, with higher T-AOC values and lower EC50 values against both ABTS+ and DPPH radicals. Fusarium sp. (HoV8) showed excellent radical-scavenging activity against DPPH, which was better than BHT.
Figure 3. ABTS+ radical-scavenging activity of the ethanol extracts of endophytic fungi, and the positive controls BHT. 
3.3. Determination of total phenol content
The standard curve of gallic acid was drawn by taking the absorbance (A) as the ordinate and the gallic acid concentration (C) as the abscissa (Fig. 4), A = 9.5268C + 0.1383, R2 = 0.9983, linear range: 13.6–68 μg/mL.
Table 2 lists the total phenolic contents of the crude extracts from 13 types of endophytic fungi (HoV1–HoV13). The total phenolic contents varied from 0.94±0.01 to 2.41±0.01 mg/g of dry weight. The highest concentration of phenols was found in the extract of HoV4 (2.41±0.01 mg/g), followed by HoV2 (2.35±0.04 mg/g), HoV12 (1.92±0.02 mg/g) and HoV1 (1.90±0.03 mg/g). In contrast, the content of HoV13 was the lowest one.  
Figure 4. Calibration curve of gallic acid. 

4. Discussion

   In the present study, the diversity study of the endophytic fungi from Hosta. Ventricosa was performed, leading to the isolation of 13 types of endophytic fungi from six genera Fusarium sp, Ilyonectria sp, Colletotrichum, Chaetomium sp, Epicoccum, Rhizopycni. Moreover, the antioxidant capacities of the ethanol extracts were assessed by T-AOC, DPPH and ABTS methods. The ethanol extracts of Fusarium sp. (HoV1) and Epicoccum. (HoV4) showed stronger comprehensiveantioxidant activities, in which higher phenolic contentswere found. It was implied that the antioxidant activitiesof those two endophytic fungi might be mainly attributed to their phenolic contents, which was consistent with the early study[20]. Otherwise, Fusarium sp. (HoV8) with moderate phenol contents showed excellent radical-scavenging activity against DPPH, suggesting that the phenol compounds were not the only source for its antioxidant activity.

5. Conclusions
In the present study, Fusarium sp. (HoV1, HoV8) and Epicoccum. (HoV4) were isolated fromHosta. Ventricosa,which are potential antioxidant agents. To the best of our knowledge, this was the first study on the antioxidant activities of the endophytic fungi from Hosta. Ventricosa. Further work on these fungi will be useful in developing new natural antioxidant agents.
This work was financially supported by National Natural Science Foundation of China (Grant No. 81703387) and the Foundation for the demonstration of tea-fruit-medicine organic ecological safety belt in Ya’an.
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陈楠, 梁晓霞*
四川农业大学 动物医学院天 然药物研究中心, 四川 成都 611130       
摘要: 以紫花玉簪为研究对象, 从其根部分离得到13株内生真菌, 并通过IST序列扩增后18S rDNA进行鉴定。以总抗氧化能力, DPPH自由基清除能力, ABTS+在自由基清除能力为指标, 研究13种内生真菌菌丝体醇提物抗氧化活性, 并测定其总多酚含量。结果表明, HoV1 (尖孢镰刀菌属)HoV4 (附球菌属)粗提物多酚含量较高(1.90±0.03 mg/g, 2.41±0.01 mg/g),且表现出较强的综合抗氧化能力。其总抗氧化能力分别为(63.46±0.26 U/g, 66.05±0.71 U/g), ABTS+自由基清除IC50分别为(30.25±0.05 μg/mL, 24.66±0.65 μg/mL), DPPH自由基清除IC50分别为(83.42±0.24 μg/mL, 59.136 μg/mL)。另外, 尖孢镰刀菌属的HoV8具有优于BHTDPPH自由基清除活性(IC50 = 16.02 μg/mL)。这些真菌都有望开发成为新型天然抗氧化制剂。 
关键词: 紫花玉簪; 内生真菌; 抗氧化活性; 总多酚含量
Received: 2018-05-31, Revised: 2018-06-19, Accepted: 2018-07-06.
Foundation items: National Natural Science Foundation of China (Grant No. 81703387) and the Foundation for the demonstration of tea-fruit-medicine organic ecological safety belt in Ya’an.
*Corresponding author. Tel.: +86-028-86291470, E-mail:  

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