Fitoterapia 87 (2013) 43–48

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Fitoterapia

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In vivo effects on the intestinal microflora of alkekengi var. francheti extracts

Xinli Li, Cuili Zhang, Weiling Li, Dachang Wu, Jianjun Liu, Li Tang, Yi Xin ⁎

Department of Biotechnology, Dalian Medical University, Dalian China article info abstract

Article history: This study aimed to investigate the effects on the intestinal microflora balance of Physalis Received 6 December 2012 alkekengi var. francheti extracts (PE) using in vivo mouse model. Luteolin-7-O-β-glycoside, Accepted in revised form 15 March 2013 Physalin J, Physalin D, and Physalin P were isolated from PE extracts and identified. Bacteroides, Available online 2 April 2013 Lactobacillus, Helicobacter, Prevotella, Odoribacter and Oribacterium were detected as dominant organisms in the intestinal tract of mice by denaturing gradient gel electrophoresis (DGGE) Keywords: analysis. The quality and quantity of Lactobacillus genus increased significantly with increasing Physalis alkekengi var. francheti concentration of PE. This study shows that the intestinal microflora balance could be improved Isolation by PE, and thus, it has the significant potential to be used as a natural agent for restoring the Intestinal microflora balance intestinal microflora balance. DGGE Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.

1. Introduction analysis to give insights into the microbial similarity and diversity of the sites, while UPGMA dendrogram construc- Physalis alkekengi var. francheti of the family of tion [8,9] and sequencing [10] were done to test for DGGE is a well-known edible and medicinal in the northeast- motifs and taxa. The results clearly indicated that the intestinal ern part of China. Its fruit calyx has been used in traditional microflora balance was improved, and therefore, P. alkekengi Chinese medicine as a therapeutic agent to remove heat, detoxify var. francheti has a significant potential to be used as a natural the body and relieve sore-throat [1].Ithasgreatmedicinal agent for improving the intestinal microflora balance. potential, and a stable form of P. alkekengi var. francheti product could be developed to fulfill the health food market. Studies on P. alkekengi var. francheti have attracted more attention in recent 2. Materials and methods years due to its potential biological functions. In addition its active components, such as physalins, polysaccharides and 2.1. Materials and chemicals flavones have been widely studied [2–6]. However, to the best of our knowledge, no studies on effects P. alkekengi var. francheti was purchased from Dalian of P. alkekengi var. francheti on the intestinal microflora balance Traditional Chinese Medicine Market, China in October 2011 and have been carried out. We therefore specifically evaluated the identified according to the identification standard of the People's effects of PE on intestinal bacterium in vitro and in vivo.PEwas Republic of China's Pharmacopeia. A voucher specimen (No. extracted with EtOH, refined by macroporous resin and analyzed LP201102) is deposited in the Department of Biotechnology, by HPLC. It showed stimulative effect on the growth of probiotics Dalian Medical University, China. AB-8 macroporous adsorptive but inhibitory effect on the pathogenic bacterium [7].Denaturing resin was purchased from the chemical plant of Nankai Uni- gradient gel electrophoresis (DGGE) was combined with image versity, China. E.Z.N.A® Stool DNA Kit was purchased from Omega (Bio-tek, USA). ExTaq polymerase was purchased from TaKaRaBiotechnologyCo.,Ltd.(Dalian,China).Polymerase ⁎ Corresponding author. Tel./fax: +86 411 86110296. Chain Reaction primers GC-357f, 518r and 357f (Table 1)were E-mail address: [email protected] (Y. Xin). synthesized by TaKaRa Biotechnology Co., Ltd. LiZhu ChangLe

0367-326X/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.03.018 44 X. Li et al. / Fitoterapia 87 (2013) 43–48 capsule (Approval Number: S10960040) and levofloxacin standardized conditions at a temperature of 22–24 °C, and capsule (Approval Number: H20066206) were purchased 20% humidity with a 12 h light/dark cycle, and they had free from Dalian Pharmacy of China. All other chemical reagents access to standard diet and water ad libitum. They were used were analytical grade. allowed to acclimatize for five days before the experiments were started. 2.2. Extraction and isolation 2.4.2. Antibiotic-induced intestinal microflora imbalance model The air dried P alkekengi var. francheti (3 kg) were extracted in mice two times in refluxing EtOH (70%) for 2 h at 60 °C. The solvent Intestinal microflora imbalance was induced by intragastric was evaporated to dryness, and the dry residue (1.5 kg) was (i.g.) administration with levofloxacin at the dose of 65 mg/kg subjected to AB-8 macroporous adsorptive resin (12 kg), eluted dissolved in water [11]. with water (14 L), 50% EtOH (22 L), and 95% EtOH (15 L) successively to yieldthreefractions(H2O fr., 50% fr. and 95% fr.). 2.4.3. Experimental design 50% fr. was collected and dried to form PE (yield, 165.0 g). Eighty mice were randomly divided into eight groups PE was further fractionated by column chromatography on (ten mice in each group). silica gel eluting with CH2Cl2/MeOH (10:1 → 1:1, v/v) to give seven fractions (SG fr. 1– 7). SG fr. 2 (20.3 g) was recrystallized Group 1 Normal control (NC), normal mice treated with water. from MeOH to give 4 (0.32 g). SG fr. 7 (48.0 g) was recrystallized Group 2 Levofloxacin control (LC), normal mice treated with from MeOH to give 1 (2.02g).SGfr.4(33.8g)wasseparatedby levofloxacin. column chromatography on silica gel eluting with CH2Cl2/MeOH Group 3 PE-L, levofloxacin-treated mice with 80 mg/kg of PE. (8:1 → 1:1, v/v) to give four fractions (SG fr. 4.1–SG fr. 4.4). SG Group 4 PE-M, levofloxacin-treated mice with 160 mg/kg of fr.4.3wasfurtherseparatedbyHPLConareverse-phasecolumn PE. (10 × 250 mm with a particle size of 5 μm, Tokyo, Japan) using Group 5 PE-H, levofloxacin-treated mice with 330 mg/kg of

MeOH/H2O (4:6) to give 2 (0.51 g) and 3 (0.88 g). PE. Group 6 LZ-L, levofloxacin-treated mice with 50 mg/kg of 2.3. High performance liquid chromatography (HPLC) analysis LiZhu ChangLe capsule. Group 7 LZ-M, levofloxacin-treated mice with 100 mg/kg of The sample PE (18 mg) was dissolved in distilled water LiZhu ChangLe capsule. (2 ml) and filtered with a 0.22 μm filter. Then the flow-through Group 8 LZ-H, levofloxacin-treated mice with 200 mg/kg of solution was analyzed by HPLC system (LC-2010A, Shimadzu, LiZhu ChangLe capsule. Japan) with a reversed-phase Waters Symmetry® C18 column (4.6 × 150 mm with a particle size of 5 μm). The mobile phase Every drug (or water for NC) was administered by i.g. once consisted of solvent A (acetonitrile) and solvent B (0.05% a day. The consumption of diet and water, and body weight phosphoric acid) with gradient program of 0–15 min, 5–8% A; were recorded daily. After 7 consecutive days of treatment, 15–25 min, 8–18% A; and 25–60 min, 18–35% A. The flow rate fecal were collected and preserved at −80 °C. was maintained at 1.0 ml/min and the injection volume was 10 μl. Ultraviolet detection was carried out at 230 nm. The 2.4.4. Deoxyribonucleic acid (DNA) extraction column temperature was kept at 35 °C. DNA was extracted from fecal samples with E.Z.N.A® Stool Compounds 1 (3.3 mg), 2 (3.08 mg), 3 (4.02 mg), and 4 DNA Kit in accordance with the manufacturer's instructions. (4.0 mg) were dissolved in distilled water (1 ml for each), and The amount and quality of DNA extracts were analyzed by filtered with a 0.22 μm filter. They were analyzed through the electrophoresis of 1% agarose gel containing ethidium bromide, same HPLC conditions as outlined above. andcomparedtoamolecularweightstandard(1kb).TheDNA concentration was measured spectrophotometrically using 2.4. In vivo effects on the intestinal microflora Biophotometer plus (NanoVue, USA) and DAN extracts were preserved at −20 °C until used. 2.4.1. Animals Female BALB/c mice weighing 20 ± 2 g, purchased from 2.4.5. Polymerase chain reaction (PCR) amplification Animal Experimental Center of Dalian Medical University Primers GC-357f and 518r were used to amplify the V3 [Certificate of quality number: SCXK (Liao) 2008-0002] were region of bacterial 16S rRNA. PCR amplification was performed used for all animal experiments. The mice were kept under with an automated thermocycler (Thermo, USA) as follows: 3 μl purified genomic DNA as template (300 ng approximately), 10 × ExTaq buffer (Mg2+ plus) 2.5 μl, dNTP mixture 4 μl, BSA Table 1 (1 mg/ml) 2.5 μl, 10 pmol of each primer, 1.25 U of ExTaq Nucleotide sequences of primers to be used in this study. polymerase, and filled up to a volume of 25 μl with sterile Primer Sequence 5′–3′ Milli-Q water. The thermal program consisted of an initial denaturation step of 94 °C for 5 min, followed by 30 cycles of GC-357fa CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGG ACGGGGGGCCTACGGGAGGCAGCAG 94 °C, 54 °C, and 72 °C for 30 s each, in which the annealing 518rb ATTACCGCGGCTGCTGG temperature was 72 °C for 7 min [12].Amplificationproducts 357fa CCTACGGGAGGCAGCAG were analyzed first by electrophoresis of 1% agarose gel a Forward. containing ethidium bromide, and compared to a molecular b Reverse. weight standard (100 bp). X. Li et al. / Fitoterapia 87 (2013) 43–48 45

2.4.6. DGGE analysis intestinal microflora balance in vivo. The chemical structures of DGGE were performed using D-Code™ Universal Mutation the isolated compounds were elucidated as Luteolin-7-O-β- Detection System (Bio-Rad, Hercules, CA). The PCR products glycoside (1), Physalin J (2), Physalin D (3), and Physalin P (4)by were electrophoresed on 8% polyacrylamide (acrylamide/ comparison of the spectral data with reported data [17–20] bisacrylamide, 37.5:1) gels containing a linear denaturant (Fig. 1). gradient ranging from 25% to 50%, with 100% denaturant defined as a solution of 7 M urea and 40% (v/v)deionized 3.2. HPLC analysis of PE formamide. Electrophoresis was performed, first for 10 min at 200 V, and subsequently for 16 h at 70 V in a 1 × TAE buffer at As it is known, flavonoid glycosides and physalins were main a constant temperature of 60 °C [13].Gelswerestainedwith active compounds in P. alkekengi var. francheti.Theycouldbe

AgNO3 [14]. purified using macroporous resin chromatography techniques. Stained gels were analyzed with Quantity One 4.6.2 gel The peaks of Luteolin-7-O-β-glycoside, Physalin J, Physalin D, analysis software (Bio-Rad). Similarities were displayed and Physalin P were at 26.2 min, 36.3 min, 41.1 min, and graphically as a dendrogram. The clustering algorithms used to 52.0 min, respectively (Fig. 1b, c, d, e). In addition in the profile calculate the dendrograms were an unweighted pair group of PE (Fig. 1a), there are also four main peaks appeared at method with arithmetic average (UPGMA) [15]. 26.2 min, 36.3 min, 41.1 min, and 52.0 min, which could be The Shannon–Wiener index of diversity (H′) [16] was regarded as Luteolin-7-O-β-glycoside, Physalin J, Physalin D, and used to determine the diversity of the bacterial community. Physalin P, respectively. Some unknown peaks before the peak of This index was calculated by: Luteolin-7-O-β-glycoside could be attributed to other flavonoid glycosides and some peaks occur with retention times beyond ′ ¼ −∑ðÞðÞ H pi lnpi 35 min could be partly attributed to other physalins. These where, pi was the proportion of the bands in the track and results showed that the extraction and isolation procedures was calculated as follows: could well give flavonoid glycosides and physalins, which also were the main components of PE. ¼ =∑ pi ni ni where, ni was the average density of peak i in the densitometric 3.3. DGGE analysis curve. The evenness (E) which reflected uniformity of bacterial The dominant intestinal microflora of NC LC, PE-L, PE-M, distribution was also computed. This index was cal- PE-H, LZ-L, LZ-M, and LZ-H were examined by DGGE analysis culated by: with universal primers targeting the V3 region of the 16S rRNA (Fig. 2a). Lane 1 was the sample from normal group, while lane E ¼ H′=ln S; 2 represented those from levofloxacin-treated group, lanes 3,4 where, S was the number of bands. and 5 represented levofloxacin-treated mice with 80, 160, and 330 mg/kg of PE respectively, and lanes 6, 7 and 8 represented 2.4.7. Sequence analysis levofloxacin-treated mice with 50, 100, and 200 mg/kg of To identify some separated and strong bands, a sterile scalpel LiZhu ChangLe capsule respectively. Obviously, F existed in NC was used to cut out the bands from the polyacrylamide gel. Gel and PE administration groups, but decreased remarkably in LC fragments were eluted in 20 μl sterile water at 4 °C overnight. groups. C existed remarkably in NC and LZ-H groups, but 4 μl of the eluted DNA was reamplified by PCR with same disappeared in other groups. I existed in LZ administration program described above, except that the forward primer was groups and U existed in NC and PE administration groups, the 357f. Each PCR product was further subjected to DGGE analysis above two bacteria (I and U) all disappeared in LC group. B only to confirm whether the band was purified or not. Subsequently, existed in LC group. Other bands almost existed in all groups idiographic sequences of the bands were obtained by TaKaRa and their intensity which is in the same position but in different Biotechnology (Dalian) Co., Ltd. Finally, the sequences were groups was similar. compared directly with those in GenBank by Blast search (NCBI). Clustering analysis based on the values of Dice coefficients was visualized in an UPGMA dendrogram to study the general 2.5. Statistical analysis patterns of community similarity among different groups by Quantity One software. Fig. 2b displayed that different groups DGGE analysis experiments were repeated at least thrice. formed the statistically significant clustering profiles. There The statistical software SPSS version 17.0 was used for analysis. were four main clusters in the dendrogram, the first was lane 2 P values were determined using the t test, P value b 0.01 was related to LC group, the second was lanes 3 and 4 related to considered significant. PE-L and PE-M groups, the third was lanes 6 and 7 related to LZ-L and LZ-M groups, and the last cluster was lanes 1, 5 and 8 3. Results and discussion related to NC, PE-H and LZ-H groups. The similarity among LC group and others was low because the intestinal microflora 3.1. Extraction and isolation of PE balance of mice was disordered by levofloxacin. In addition, NC, LZ-H and PE-H groups shared a relatively high similarity. P. alkekengi var. francheti was extracted by 70% EtOH DGGE profiles displayed the typical characteristics of general refluxing with a yield of 20.0%. After fractionated by AB-8 bacteria in the intestinal tract. Each band derives possibly from macroporous adsorptive resin, 50% fr. with a yield of 5.5% one phylogenetically distinct community, hence, an estimation was isolated for further analysis of the effects on the of species number could be based on the total number of the 46 X. Li et al. / Fitoterapia 87 (2013) 43–48

Fig. 1. A preliminary assay for the components of PE by HPLC. 1: Luteolin-7-O-β-glycoside; 2: Physalin J; 3: Physalin D; 4: Physalin P. (a): The HPLC profile of PE; (b): The HPLC profile of Luteolin-7-O-β-glycoside; (c): The HPLC profile of Physalin J; (d): The HPLC profile of Physalin D; (e): The HPLC profile of Physalin P.

bands in the profile [21]. H′ reflecting the structural diversity of Table 3 shows result for the closest relatives based on results the bacterial community [16] was calculated on the basis of the of BLAST searches with DNA sequences obtained from DGGE number and relative intensities of bands on the gel (Table 2). gel bands and identified by cluster analysis. Bands in the same It was clearly shown that diversity in LC, PE-L and LZ-L position but in different lanes were excised and sequenced to groups, vis-a-vis NC group, decreased with statistical signifi- confirm that they had the same identity (data not shown). C, F, I cance. Compared to NC group, the number of bands was richer in and U were sequenced and identified as Lactobacillus johnsonii, PE-M, PE-H and LZ-H groups. Lactobacillus gasseri, Lactobacillus amylolyticus and Lactobacillus

Fig. 2. Representative DGGE profiles (a) and UPGMA dendrograms (b) of different groups. 1: Normal control (NC), normal mice treated with water. 2: Levofloxacin control (LC), normal mice treated with levofloxacin. 3: PE-L, levofloxacin-treated mice with 80 mg/kg of PE. 4: PE-M, levofloxacin-treated mice with 160 mg/kg of PE. 5: PE-H, levofloxacin-treated mice with 330 mg/kg of PE. 6: LZ-L, levofloxacin-treated mice with 50 mg/kg of LiZhu ChangLe capsule (Live Bifidobacterium preparation). 7: LZ-M, levofloxacin-treated mice with 100 mg/kg of LiZhu ChangLe capsule. 8: LZ-H, levofloxacin-treated mice with 200 mg/kg of LiZhu ChangLe capsule. X. Li et al. / Fitoterapia 87 (2013) 43–48 47

Table 2 and might be related to the bacterial death in the tracts of a Microflora diversity index analysis ðÞx s; n ¼ 10 . levofloxacin-treated mice. The bacterial composition and

Group S H′ E structure were divided into four clusters as distinct groups. The intestinal microflora communities of NC, LZ-H and PE-H NC 17.6 ± 2.6 2.7787 ± 0.0456 0.9355 ± 0.0039 ⁎⁎ ⁎ groups displayed a relatively high homology. That is to say, LC 13.1 ± 0.7 2.3711 ± 0.0637 0.9390 ± 0.0042 ⁎⁎ ⁎ PE-L 13.7 ± 1.1 2.2819 ± 0.0778 0.8896 ± 0.0303 the intestinal microflora imbalance could be improved by PE, ⁎⁎ PE-M 20.4 ± 0.8 2.5368 ± 0.1556 0.8403 ± 0.0611 and the effect of PE is similar to that of LiZhu ChangLe capsule ⁎ PE-H 20.2 ± 0.4 2.5889 ± 0.1221 0.8714 ± 0.0305 (positive drug). The results from sequencing provided more ⁎⁎ ⁎ LZ-L 13.5 ± 1.2 2.5005 ± 0.0650 0.9763 ± 0.0162 ⁎⁎ precise information to confirm the intestinal bacterial compo- LZ-M 15.0 ± 0.8 2.6032 ± 0.0773 0.9613 ± 0.0286 ⁎ LZ-H 20.8 ± 1.0 2.7751 ± 0.0707 0.9188 ± 0.0129 sition of the different groups. The bacteria from the genera of Bacteroides, Lactobacillus, Helicobacter, Prevotella, Odoribacter a ⁎⁎ ⁎ Compared to the normal group P b 0.01, Pb0.05. and Oribacterium were dominant organisms in the intestinal tract of mice by DGGE analysis. Presumably, the transformation reuteri with the similarity of 97%, 100%, 98% and 91%, res- of dominant organisms could be the risk factors associated with pectively, all belonging to Lactobacillus genus. L. johnsonii existed side effects of antibiotics, specifically, Lactobacillus decreased in in NC and LZ-H groups, L. gasseri, L. reuteri and L. amylolyticus levofloxacin-treated group and turned into pathogenic bacte- increased in PE and LZ groups, while there was nearly no band rium. Moreover, with the dose of PE increased, Lactobacillus at the corresponding position of LC group particularly. A, B, D, S, increased remarkably both in terms of quality and quantity. T and V were sequenced and identified as Bacteroides ovatus, The growth of Lactobacillus could be promoted by PE in vivo Bacteroides sp., Bacteroides sp., Bacteroides sp., Bacteroides oral which was consistent with the in vitro results [7]. taxon and Bacteroides coprosuis with the similarity of 99%, 100%, The abuse of antibiotics has become a world-wide public 96%, 96%, 98% and 98%, respectively, all belonging to Bacteroides health problem because the inappropriate use of antibiotics is a genus. B. ovatus and Bacteroides sp. increased remarkably major cause of the development of antibiotic-resistant bacterial in LC group. L, N, O, P and Q were sequenced and identified strains [22]. Meanwhile, side effects affecting the gastrointes- as Prevotella micans, Prevotella dentails, Prevotella amnii, tinal tract are common, such as abdominal pain, diarrhea, and Prevotella buccae and Prevotella sp. oral taxon with the astriction [23,11]. Non-pathogenic diarrhea is caused by an similarity of 98%, 97%, 99%, 94% and 96%, respectively, all imbalance of intestinal microflora in most cases, thus, once the belonging to Prevotella genus. E, G, H and K were sequenced intestinal microflora balance is disturbed, pathogenic bacteria, andidentifiedasHelicobacter pullorum, Helicobacter bilis, viruses, and coccidia can easily infect the host. Helicobacter cinaedi and Helicobacter hepaticus with the We have regarded flavonoid glycosides (Luteolin-7-O-β- similarity of 99%, 95%, 96% and 97%, respectively, all belonging glycoside) and physalins (Physalin J, Physalin D, Physalin P), to Helicobacter genus. R and J were sequenced and identified as which were the main components of P. alkekengi var. francheti Odoribacter splanchnicus, Oribacterium sp. with the similarity of by HPLC analysis, as the active compounds having promoting 96% and 80%, respectively. effects on Lactobacillus in vitro [7] and in vivo. Imbalance in DGGE analysis indicated that levofloxacin has an impor- intestinal microflora caused by antibiotics could induce tant effect on the intestinal microflora of mice. A decreased pathogenic diseases. It is suggested that an improvement in diversity of bacterium composition in LC group was found the intestinal microflora, that is, an increase in the amount of

Table 3 Sequences of PCR amplicons derived from DGGE gels and identities based on the BLAST database.

Selected band Most similar sequence relative Bacteria genus Identity (%) (GenBank accession number)

A Bacteroides ovatus (AAXF02000050.1) Bacteroides 99 B Bacteroides sp. (ADCL01000128.1) 100 D Bacteroides sp. (ACRQ01000064.1) 96 S Bacteroides sp. (ACTC01000133.1) 96 T Bacteroides oral taxon (ADCM01000022.1) 98 V Bacteroides coprosuis (AFFW01000002.1) 98 C Lactobacillus johnsonii (AFQJ01000002.1) Lactobacillus 97 F Lactobacillus gasseri (ADFT01000001.1) 100 I Lactobacillus amylolyticus (ADNY01000006.1) 98 U Lactobacillus reuteri (CACS02000061.1) 91 E Helicobacter pullorum (ABQU01000097.1) Helicobacter 99 G Helicobacter bilis (ACDN01000023.1) 95 H Helicobacter cinaedi (ABQT01000054.1) 96 K Helicobacter hepaticus (NC_004917.1) 97 L Prevotella micans (AGWK01000061.1) Prevotella 98 N Prevotella dentails (AFPW01000057.1) 97 O Prevotella amnii (ADFQ01000002.1) 99 P Prevotella buccae (AEPD01000042.1) 94 Q Prevotella sp. oral taxon (ACZS01000106.1) 96 R Odoribacter splanchnicus (NC_015160.1) Odoribacter 96 J Oribacterium sp. (ACIQ02000009.1) Oribacterium 80 48 X. Li et al. / Fitoterapia 87 (2013) 43–48

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