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Planta Med 2022; 88(03/04): 254-261
DOI: 10.1055/a-1647-2866
Biological and Pharmacological Activity
Original Papers

Gut Microbiota of Pigs Metabolizes Extracts of Filipendula ulmaria and Orthosiphon aristatus–Herbal Remedies Used in Urinary Tract Disorders

Dominik Popowski 
1   MicrobiotaLab, Department of Pharmacognosy and Molecular Basis of Phytotherapy, Centre for Preclinical Studies, Medical University of Warsaw, Warsaw, Poland
,
Jürgen Zentek 
2   Institute of Animal Nutrition, Freie Universität Berlin, Berlin, Germany
,
Jakub P. Piwowarski 
1   MicrobiotaLab, Department of Pharmacognosy and Molecular Basis of Phytotherapy, Centre for Preclinical Studies, Medical University of Warsaw, Warsaw, Poland
2   Institute of Animal Nutrition, Freie Universität Berlin, Berlin, Germany
,
Sebastian Granica 
1   MicrobiotaLab, Department of Pharmacognosy and Molecular Basis of Phytotherapy, Centre for Preclinical Studies, Medical University of Warsaw, Warsaw, Poland
› Author Affiliations

Supported by: Narodowe Centrum Nauki 2018/29/B/NZ7/01873
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Abstract

Urinary tract infections influence the mortality rate in pigs and are linked to extensive antibiotic usage in the farm industry. Filipendula ulmaria (L.) Maxim. and Orthosiphon aristatus (Blume) Miq. are widespread medicinal plants traditionally used to treat urinary tract disorders. As their preparations are orally administered, the metabolism of their constituents by gut microbiota before absorption should be considered. Until now, no experiments had been performed to describe the biotransformation of tthose plantsʼ extracts by animal gut microbiota. The study evaluates the influence of pig intestinal microbiota on the structure of active compounds in flowers of F. ulmaria and leaves of O. aristatus. The incubations of the extracts with piglet gut microbiota were performed in anaerobic conditions, and the samples of the batch culture were collected for 24 h. In F. ulmaria, the main metabolites were quercetin and kaempferol, which were products of the deglycosylation of flavonoids. After 24 h incubation of O. aristatus extract with the piglet gut microbiota, 2 main metabolites were observed. One, tentatively identified as 3-(3-dihydroxyphenyl)propionic acid, is likely the primary metabolite of the most abundant depsides and phenolic acids. The results confirm the formation of the compounds with anti-inflammatory and diuretic activity in the microbiota cultures, which might suggest F. ulmaria and O. aristatus for treating urinary tract disorders in piglets. Based on the similarities of human and pig gut microbiota, the pig model can help estimate the metabolic pathways of natural products in humans.


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Key words

Filipendula ulmaria - Orthosiphon aristatus - Rosaceae - Lamiaceae - microbiota - metabolism - swine, piglets - urinary tract

Abbreviations

BHI: brain heart infusion
FS: fecal slurries
FUX: Filipendulae ulmariae flos water infusion
OAX: Orthosiphonis folium water infusion
UPEC: uropathogenic Escherichia coli
UTI: urinary tract infections
 

Introduction

Filipendula ulmaria (L.) Maxim. (previously Spiraea ulmaria L.), commonly known as meadowsweet, is an herbaceous plant material belonging to the Rosaceae family [1] that is traditionally used in humans for colds, heartburn, arthritis, and UTI [2]. Nowadays, it is considered an antipyretic, analgesic, and antacid used to treat kidney and bladder diseases and gastric ulceration. Due to the attributed properties, meadowsweet is also widely used in veterinary medicine, suggested as a spring tonic for animals [3]. Because of its anti-acid potential, it is also recommended to treat performance horses suffering from gastric ulceration, gastritis, and colitis [4]. The phytochemical studies focused on the chemical composition of extract prepared from flowers of F. ulmaria, which is contemporarily used in medicine, showed that it contains flavonoids, tannins, phenolic glycosides, and essential oil [5], [6], [7].

Orthosiphon aristatus (Blume) Miq. (syn. Orthosiphon stamineus Benth.), also called Java tea or catʼs whiskers, is an herbaceous plant material from the Lamiaceae family. It occurs in Euroasia, Australia, and the Pacific region [8] and is used in Malesia and other countries as a food beverage in the form of herbal tea [9]. Java tea leaves are a popular remedy for treating eruptive fever, epilepsy, gallstone, hepatitis, rheumatism, hypertension, syphilis, gonorrhea, and renal calculus. However, the most important and well-documented activity of Orthosiphon is a diuretic effect in humans and animals [10]. Extracts prepared from this plant material contain mostly phenolics, including flavonoids and caffeic acid derivatives. Other constituents of Java tea are isopimarane-type diterpenes, staminane-type diterpenes, and pentacyclic triterpenes such as betulinic acid, oleanolic acid, and ursolic acid [8]. On the market, several veterinary products containing Java tea leaves or extract are used to support weight loss by cats and dogs and as a detoxifying or lung-protecting agent for horses [11], [12], [13].

UTIs have been reported as a primary cause of death of pigs [14]. Cystitis and pyelonephritis were also a leading cause of death in a multi-herd prospective study [15] and the most common secondary lesions observed in a large herd [16]. The investigation of UPEC strains causing porcine pyelonephritis indicated that the examined strains belonged to clonal groups A and B1, typically considered to be multi-drug resistant [17]. Despite regulations, extensive antibiotic usage in pig production connected with rising anti-microbial resistance is still an issue [18], creating a need for alternative approaches. One considered strategy is using medicinal plant extracts, which have already been used in commercial supplementary products for UTI prevention in pigs [19]. Meadowsweet and Java tea are medicinal plants widespread in their areas of distribution [1], [8], so they could be an easily accessible resource in countering UTIs in pigs.

Gut microbiota is a complex organ present in the gastrointestinal tract of humans and animals. It takes part in many crucial processes such as digestion, immunomodulation, and metabolism of xenobiotics [20]. Several studies showed that the microbiome could selectively transform natural products contained in plant materials used as food products or herbal remedies [21], [22]. It was proven that plant flavonoids, procyanidins, or ellagitannins could be metabolized by specific bacteria and fungi present in the human gut to smaller bioavailable organic compounds [23]. However, few studies have focused on the metabolic role of the animal microbiome in the context of the use of medicinal plant materials in veterinary medicine. [24], [25]. Additionally, the pig gastrointestinal tract model is being tested in studies assessing human gut metabolism studies. Therefore, improvements such as human gut microbiota transplantation into germ free pigs are being developed to overcome the differences in bacterial composition between species [26].

The present research aimed to establish the influence of swine microbiota on the chemical composition of extracts prepared from meadowsweet and Java tea–medicinal plant materials commonly used in animal medicine and nutrition. The changes in the extract composition after incubation with piglet gut microbiota ex vivo were investigated using the UHPLC-DAD-MSn approach. The obtained results were compared to previous reports that focused on human microbiota metabolism.


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Results and Discussion

FUX and OAX were obtained in a yield of 59.20 and 19.36% (relative to the starting material). The results of the phytochemical analysis of FUX used in the experiments were described and discussed previously [5]. The UHPLC-DAD-MSn analysis of the OAX ([Fig. 1]; [Table 1]) shows that the main plant specialized metabolites in boiling water extract of Orthosiphonis folium were phenolic acids and depsides, with rosmarinic acid (18) as the most abundant constituent ([M – H]− m/z 359, [M – Caffeoyl – H2O]− m/z 161). In comparison to previously examined methanol-water (1 : 1, v : v) extract [27], significantly lesser amounts of sagerinic acid (20), salvianolic acid B (21), and salvianolic acid B dehydroxylated derivative (22) were present in OAX. Sagerinic acid (20) was identified based on the loss of 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoyl moiety and cleavage of cyclobutene ring ([M – H]− m/z 719, [M-C9H10O5-H] m/z 521, [M – C9H9O5 – C9H6O3 – H]− m/z 359) [28]. Similar deconjunction of the 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoyl moiety occurred in the case of salvianolic acids A (19, [M – H]− m/z 493, [M – C9H10O5 – H] m/z 295) and B (21, [M – H]− m/z 717, [M – C9H10O5 – H] m/z 519) and dehydroxysalvianolic acid B (22, [M – H]− m/z 701, [M – C9H10O5 – H] m/z 503). A significantly higher amount of chicoric (17, [M – H]− m/z 473, [M – Caffeoyl – H] m/z 311) acid was detected in water infusion than in the methanol-water extract. Caffeic (14) and monocaffeoyltartaric acids (15) were also detected [27]. Another phenolic acid that is of significant abundance in the extract was (R)-3-(3,4-dihydroxyphenyl)-2-hydroxypropanoic acid, also known as danshensu (13, [M – H]− m/z 197). Lithospermic acid (15) was identified based on deconjunction of the carboxyl group and 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoyl moiety ([M + H2O – H]− m/z 555, [M – H]− m/z 537, [M – COOH – H]− m/z 493, [M – COOH – C9H10O5 – H] m/z 295) [29]. The only diterpenes that were tentatively identified in the extract were orthosiphol M or I (26, [M + HCOOH – H]− m/z 615, [M – H]− m/z 569) and orthosiphol D (27, [M – H]− m/z 551, [M – CH2CO – H]− m/z 509, [M – 2(CH2CO) – H]− m/z 467) [30], [31]. Methoxyflavones–3′-hydroxy-5,6,7,4′-tetramethoxyflavone (23), sinensetin (24), and eupatorin (25)–were detected in the positive ionization mode [10].

Zoom Image
Fig. 1 Base peak chromatograms (in negative BPC [−] and positive ionization BPC [+] mode) of the raw OAX solution. Peak assignment based on reference data UV and MSn data, see [Table 1].

Table 1 Compounds detected and tentatively identified in the raw lyophilized OAX (peaks 13 – 27), the lyophilized post-incubation mixtures, obtained after the incubation of FUX (peaks 1 – 12, M1 and M2) and OAX (peaks M3 and M4) with pig gut microbiota. The m/z values obtained from the subtracted data. Peak assignment based on reference HPLC-MS data. Identification of peaks 4, 5, and 11 based on reference NMR data. b: most abundant fragmentary ion; sh: shoulder peak.

Peak No.

t R [min]

Compound identity

Spectrum max [nm]

Ionization

m/z

MSn

Constituents of Filipendula ulmaria (L.) Maxim. flowers extract (FUX)

1

21.6

monotropitin

283

(−)

491

MS2: 445, 413, 293b

2

34.7

quercetin O-hexoside

257, 353

(−)

463

MS2: 301b

3

35.8

quercetin O-hexoside

255, 353

(−)

463

MS2: 301b

4

38.3

quercetin 3-O-β-(2″-O-galloyl)-d-galactopyranoside

259, 357

(−)

615

MS2: 313, 301b

5

39.1

quercetin 3-O-β-(2″-O-galloyl)-d-glucopyranoside

264, 356

(−)

615

MS2: 463, 301b

6

39.8

isorhamnetin O-hexoside

268, 352

(−)

477

MS2: 462, 315b, 300

7

40.9

kaempferol O-hexoside

264, 344

(−)

447

MS2: 327, 285b, 255, 179

8

41.9

quercetin O-hexoside

252, 364

(−)

463

MS2: 301b

MS3: 273, 179b, 151

9

42.3

isorhamnetin O-hexoside

267, 59

(−)

477

MS2: 357, 315b, 300, 271

10

42.7

kaempferol O-hexoside

265, 362

(−)

447

MS2: 327, 285b, 151

MS3: 257, 241, 151b, 107

11

44.8

kaempferol 3-O-β-(2″-O-galloyl)-d-glucopyranoside

266, 351

(−)

599

MS2: 313, 285b

12

48.5

acylated isorhamnetin O-hexoside

354

(−)

519

MS2: 315b, 300, 191

Metabolites of FUX constituents

M1

54.1

quercetin

255, 368

(−)

301

MS2: 257, 179b, 151

M2

64.6

kaempferol

263, 362

(−)

285

MS2: 241, 185, 151b

Constituents of Orthosiphon aristatus leaves extract (OAX)

13

8.8

(R)-3-(3,4-dihydroxyphenyl)-2-hydroxypropanoic acid

279

(−)

197

MS2: 179

14

14.1

caffeic acid

298(sh), 326

(−)

179

–

15

14.2

monocaffeoyltartaric acid

298(sh), 326

(−)

311

MS2: 267, 179, 149b

16

38.3

lithospermic acid

275, 323

(−)

537

MS2: 493b, 383, 295

17

39.2

chicoric acid

289(sh), 327

(−)

473

MS2: 311b, 293, 149

18

49.5

rosmarinic acid

294(sh), 327

(−)

359

MS2: 223, 197, 179, 161b

19

52.4

salvianolic acid A

284, 331

(−)

493

MS2: 383, 313, 295b

20

54.3

sagerinic acid

277, 318

(−)

719

MS2: 521, 359b, 341, 279

21

56.6

salvianolic acid B

285, 306, 332(sh)

(−)

717

MS2: 519b, 321

22

62.0

dehydroxylated salvianolic acid B

284, 310, 332(sh)

(−)

701

MS2: 503b, 321

23

67.4

3′-hydroxy-5,6,7,4′-tetramethoxyflavone

279, 323

(+)

359

MS2: 344, 315, 298b

24

70.0

sinensetin

268, 327

(+)

373

MS2: 358, 312b

25

71.6

eupatorin

276, 334

(+)

345

MS2: 330b, 312

26

72.3

orthosiphol M/I

–

(−)

569

–

27

74.6

orthosiphol D

331

(−)

551

MS2: 509, 467b, 309, 199

Metabolites of OAX constituents

M3

21.4

3-(3-hydroxyphenyl)propionic acid

272, 280(sh)

(−)

165

MS2: 121

M4

31.9

unidentified metabolite

279

(−)

345

MS2: 327b, 301, 257, 241

The results of the incubations of 2 mg/mL FUX ([Fig. 2]) show that the most noticeable metabolites being detected after 2 h of fermentation were aglycones: quercetin (M1) and kaempferol (M2). The aglycon level rose, which is linked to the drop in the monoglycosides of quercetin (8) and kaempferol (10) signals at the subsequent time points, as 8 and 10 were finally not detected in the sample taken after 24 h of incubation. In the previous experiments with human gut microbiota [5], a lower rate of deglycosylation was observed in the case of the galloylated derivatives of FUX, as after 24 h of incubation, no other flavonoid glycosides were observed. In the current samples prepared with pig gut microbiota, monoglycosides other than 8 and 10, galloylated monoglycosides, and isorhamnetin glucoside remained in the extract after 24 h. The results indicated a comparable order of deglycosylation but a different process yield. This might be connected with phylogenetic profile divergence between human and pig gut microbiota [26], especially within the scope of bacteria responsible for flavonoid O-deglycosylation, such as Bifidobacterium [32]. Additionally, similarly to the mentioned human gut microbiota incubations of FUX, no urolithins that are products of hydrolyzable tannins fraction metabolism were detected after 24 h. The experiments performed using higher FUX concentration (6 mg/mL) did not significantly differ in the metabolism outcome, as only M1 and M2 were detected. However, the order of glycosylation rate was not visible, as no 8 and 10 signal drops in reference to other flavonoid glycosides were observed (data not shown). The deglycosylation of monotropitin (1, [M + HCOOH – H]− m/z 491, [M – H]− m/z 445), the most abundant and the only confirmed salicylic glycoside of FUX, was not observed [33].

Zoom Image
Fig. 2 UV (254 nm) chromatograms of the samples of the incubation mixture. 2 mg/mL FUX incubated with the pig gut microbiota (FS). Samples collected at 0, 2, 4, 6, and 24 h. Peak assignment based on UV and MSn data, see [Table 1].

In the case of OAX, the subtraction process did not clarify the results sufficiently enough to confirm visible differences in 2 and 6 mg/mL incubation outcomes. Thus, when using an OAX concentration of 6 mg/mL, signals were of higher intensity and easier to distinguish. Thus, this variant was used to evaluate the OAX constituentsʼ biotransformation (for 2 mg/mL, data not shown). No significant change in the constitution occurred in the first 6 h of incubation ([Fig. 3]). After 24 h, the phenolics were not detected in the sample, and the only remaining constituents of the raw OAX were diterpenes (26, 27). The two most abundant metabolites were M3, tentatively identified as 3-hydroxyphenylpropionic acid ([2 M – H]− m/z 331, [M – H]− m/z 165) and M4 (unidentified metabolite; [M – H]− m/z 345). M3 was likely the product of phenolic acids and depsides biotransformation, as it is a common, well-described product from the metabolism of phenolics by mammalian gut microbiota [34], [35]. Eupatorin metabolism was previously tested in vivo on rats and ex vivo on rat intestinal microbiota using a similar anaerobic fermentation method [36]. However, no matching metabolites were detected in the analyzed samples, which might be associated with different duration of incubation (samples collected only after 12 h) and microbiome composition between pig [37] and rat [38].

Zoom Image
Fig. 3 Base peak chromatograms (in negative BPC [−] and positive ionization mode BPC [+]) of the samples of the incubation mixture. 6 mg/mL OAX incubated with the pig gut microbiota (FS). Samples collected at 0, 2, 4, 6, and 24 h. Peak assignment based on UV and MSn data in reference to the raw OAX analysis, see [Table 1].

In the post-incubation mixtures, a variety of phenolic compounds of known anti-inflammatory and diuretic activities were detected [39], which, after absorption in the large intestine, may play a significant role in countering bacterial infections within the urinary tract. Additionally, biotransformation to smaller compounds might result in enhanced bioavailability [40]. Therefore, the use of F. ulmaria and O. aristatus should be considered in treating and preventing urinary tract conditions in pigs. The similarities in the biotransformation productsʼ profiles between analogous experiments with human and pig gut microbiota ex vivo indicate that the pig model can be considered useful in describing the anti-UTI performance of orally-administrated phytochemicals in humans, as its advantages over small-animal models in assessing UTI pathogenesis were already detailed [41].


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Materials and Methods

General experiment procedures

Water, used for extraction, the UHPLC phase, and dissolving extract samples, was purified with the Simplicity System (Merck KDaD). Acetonitrile and formic acid for chromatography (HPLC grade) and methanol to dissolve samples before UHPLC-DAD-MSn analysis (gradient grade) were purchased from POCh.

Meadowsweet flowers (F. ulmariae flos) were collected from the Garden of Medical Plants of the Department of Pharmacognosy, Medical University of Lublin, Poland. A voucher specimen (No. 09 – 09 – 2017_A) was deposited in the Garden of Medical Plants collection. Java tea leaves (O. folium; dried and fragmented) were purchased from Kawon, Poland (batch number, 587.2016). A voucher specimen is deposited in the Herbarium of the Department of Pharmacognosy and Molecular Basis of Phytotherapy, the Medical University of Warsaw, Poland (No. [OF587.2016]). Plant materials identities were confirmed by Prof. Sebastian Granica according to Rutkowski [42] (meadowsweet flowers) and using microscopic and TLC identification methods described in detail in the monograph of the 11th edition of Polish Pharmacopoeia (Java tea) [43].


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Preparation of aqueous extracts from F. ulmariae flos and O. folium

Two-stage extraction of the plant material was performed with boiling water with a material : solvent ratio (m/v) as follows: 1 : 16 and 1 : 10 for 15 min each stage. Fifty g of dried plant material was used. Combined extracts were filtered (Whatman qualitative paper grade 1 [GE Healthcare]) and freeze-dried. The extraction yield was 29.60 and 9.68 g for FUX and OAX, respectively. Twenty mg of lyophilized FUX and OAX were dissolved in 1 mL of water for the phytochemical characterization of the extract, filtered through a 0.45 µm PVDR syringe filter (Kinesis), and subjected to UHPLC analysis.


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UHPLC-DAD-MSn analysis of the extracts and the post-incubation mixtures

The UHPLC-DAD-MSn analysis of the freeze-dried FUX, OAX, and post-incubation mixtures was performed on a UHPLC-3000 RS system (Dionex) with DAD detection (wavelength range: 200 – 450 nm) and splitless connection with an AmaZon SL ion trap mass spectrometer with an ESI interface (Bruker Daltonik GmbH). The ESI unit was set to the following parameters: nebulizer pressure, 40 psi; drying gas flow rate, 9 L/min; nitrogen gas temperature, 300 °C; capillary voltage, 4.5 kV. The MS spectra were registered by scanning from m/z 70 to 2200. Kinetex XB-C18 columns were used (Phenomenex; 150 × 3.0 mm, 2.6 µm for the phytochemical characterization of the extracts; 150 × 2.1 mm, 1.7 µm for the post-incubation mixture analysis). The mobile phase (A) was H2O/formic acid (100 : 0.1, v/v), and the mobile phase (B) was acetonitrile/formic acid (100 : 0.1, v/v). The gradient programs and the flow rates were respectively 0 – 60 min 3 – 26% B, 60 – 80 min 26 – 90% B and 0.4 ml/min for the phytochemical characterization of the extract; 0 – 50 min 5 – 26% B, 60 – 90 min 26 – 90% B and 0.3 ml/min the post-incubation mixture analysis. The column oven temperature was set to 25 °C. The injection volume was 2 µL. Data were processed and analyzed using Bruker Data Analysis 5.3 Build 5.3.342 (Bruker Daltonik GmbH). The background subtraction was performed using the built-in Xpose algorithm (retention time window, 0.5 s; ratio, 10). The MS data of the test samples was subtracted from the results of the respective control sample analysis to remove the MS signals of the background.


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Incubation with piglet gut microbiota

The pig gut microbiota incubation experiments were performed in a Bactron 300 anaerobic chamber (Sheldon Manufacturing Inc.). A BHI was purchased from DIFCO and used as the growth medium. The feces were obtained from piglets used as a control group in another project approved by the State Office of Health and Social Affairs Berlin (LAGeSo Reg. No. A 0439/17). The piglets were kept in the pens (4 piglets per pen, 1.75 m2 per piglet) and fed with an isonutritive diet containing wheat, barley, soybean, vitamins, and minerals premix. Immediately after defecation, the feces were collected from 39 – 66-day-old piglets using sterile containers, and the samples were subsequently processed within 30 min. Feces collected from 2 different animals were subjected separately to incubation with OAX and FUX, respectively. Each experiment was performed using pig feces from 1 pig for incubation of 1 extract. BHI was prepared according to the manufacturerʼs instructions, and FS were prepared by suspending pig feces in BHI (1 : 10, w/v; 37 °C). To achieve anaerobic conditions, BHI was stored at 37 °C in the incubator inside an anaerobic chamber 48 h prior to the experiment. Extract solutions of 40 and 120 mg/ml were prepared by dissolving lyophilized extract in purified water. The incubation mixture contained 0.5 ml of extract solution, 1 ml of FS, and 8.5 ml of BHI to obtain the final 2 and 6 mg/mL extract concentration in the batch cultures. The incubations were performed at 37 °C inside an anaerobic chamber. Control samples were BHI and FS in BHI. One ml of the batch culture was collected after 0, 2, 4, 6, and 24 h, immediately frozen at − 80 °C, and freeze-dried. For the chromatographic analysis, the samples were put in an ultrasonic bath for 15 min with the addition of 1 ml of MeOH and centrifuged afterward. The resulting supernatant was filtered through a 0.45 µm PVDR syringe filter and analyzed using UHPLC-DAD-MSn.


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Contributorsʼ Statement

Data collection: D. P., J. P. P. Design of the study: J. P., J. Z., S. G. Analysis and interpretation of the data: D. P. Drafting the manuscript: D. P. Critical revision of the manuscript: J. P. P., S. G.


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Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

The project was financially supported by Polish National Science Centre research grant OPUS 15 No. 2018/29/B/NZ7/01873. This project was carried out using CePT infrastructure financed by the European Regional Development Fund within the Operational Programme “Innovative economy” for 2007 – 2013.


Correspondence

Jakub P. Piwowarski
Associate professor
Medical University of Warsaw
Department of Pharmacognosy and Molecular Basis of Phytotherapy
ul. Banacha 1
02-097 Warsaw
Poland   
Phone: + 48 2 25 72 09 53   
Fax: + 48 2 25 72 09 53   

Publication History

Received: 01 April 2021

Accepted after revision: 25 August 2021

Article published online:
08 October 2021

© 2021. Thieme. All rights reserved.

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Zoom Image
Fig. 1 Base peak chromatograms (in negative BPC [−] and positive ionization BPC [+] mode) of the raw OAX solution. Peak assignment based on reference data UV and MSn data, see [Table 1].
Zoom Image
Fig. 2 UV (254 nm) chromatograms of the samples of the incubation mixture. 2 mg/mL FUX incubated with the pig gut microbiota (FS). Samples collected at 0, 2, 4, 6, and 24 h. Peak assignment based on UV and MSn data, see [Table 1].
Zoom Image
Fig. 3 Base peak chromatograms (in negative BPC [−] and positive ionization mode BPC [+]) of the samples of the incubation mixture. 6 mg/mL OAX incubated with the pig gut microbiota (FS). Samples collected at 0, 2, 4, 6, and 24 h. Peak assignment based on UV and MSn data in reference to the raw OAX analysis, see [Table 1].