Applied Microbiology and Biotechnology
Medicinal herbs as a potential strategy to decrease methane productionby rumen microbiota: A systematic evaluationwith a focus on Perillafrutescens seedextract
Jiakun Wang1, Mei Liu1, Yuelei Wu1, Liang Wang1, Jianxin Liu1, Linshu Jiang2,Zhongtang Yu3
1IMoE Key Laboratory of Molecular Animal Nutrition, College of Animal Sciences, Zhejiang University, Hangzhou 310058, P.R. China
2Beijing Key Laboratory for Dairy Cow Nutrition, Beijing 102206, China
3Department of Animal Sciences, The Ohio State University, Columbus, USA
Table S1
Herbal plants and parts whose extracts were evaluated in thisstudy
Herbal plants / Latin names of plants / Parts / CH4productionas compared to the control*
Cortex garcinia / Garcinia mangostana L. / Pericarp / -74.86
Cortex magnoliae officinalis / Magnolia officinalisRehd. etWils. / Peels of stem, root and twig / -60.11
Olibanum / BoswelliacarteriiBirdw. / Resin / -45.36
Lignum sappan / Caesalpiniasappan L. / Heartwood / -44.81
Rhizomacurcumaelongae / Curcuma longa L. / Rhizomes / -42.62
Cortex acanthopanaxradicis / Acanthopanaxgracilistylus W. W. Smith. / Velamen / -42.62
Fructuscnidii / Cnidiummonnieri (Zhou et al.) Cuss. / Seeds / -38.80
Radix polygalae / Polygala tenuifoliaWilld. / Roots / -33.33
Fructusperillae / Perillafrutescens (Zhou et al.) Britt. / Seeds / -30.05
Flosmagnoliae / Magnolia biondiiPamp. / Flower buds / -28.96
Radix linderae / Linderaaggregata (Sims) Kosterm. / Tubers / -24.59
Rhizoma et radix notopterygii / Notopterygiumincisum Ting. ex H. T. Chang. / Rhizomes / -24.59
Cortex cinnamomi / Cinnamomum cassiaPresl. / Bark / -24.04
Rhizomazingiberispreparata / ZingiberofficinaleRosc. / Rhizomes / -23.50
Cortex albiziae / AlbiziajulibrissinDurazz. / Bark / -21.86
Rhizomazingiberis / ZingiberofficinaleRosc. / Rhizomes / -21.31
Herbacynomorii / CynomoriumsongaricumRupr. / Stems / -20.77
Fructusamomi / AmomumvillosumLour. / Fruits / -20.22
Herbaseu radix cirsiijaponici / Cirsiumjaponicum DC. / Aerial parts / -19.67
Fructusalpiniaeoxyphyllae / AlpiniaoxyphyllaMiq. / Fruits / -19.13
Radix rhapontici / Rhapontisumunflorum (Zhou et al.) DC. / Roots / -18.58
Fructusviticis / Vitextrifolia L. var. simplicifolia Cham. / Fruits / -18.03
Radix paeoniaerubra / Paeonialactiflora Pall. / Roots / -15.85
Herbageranii / ErodiumstephanianumWilld. / Aerial parts / -15.30
Fructusanisistellati / Illiciumverum Hook. f. / Fruits / -15.30
Fructusaurantiiimmaturus / Citrus aurantium L. / Fruitlets / -14.75
Herbaequisetihiemalis / Equisetum hiemale L. / Aerial parts / -14.21
Semen litchi / Litchi chinensisSonn. / Seeds / -13.66
Semen ziziphispinosae / Ziziphusjujuba Mill. var. spinosa (Bunge) Hu ex H.F. Chou. / Seeds / -12.57
Folium perillae / Perillafrutescens (Zhou et al.) Britt. / Leaves / -12.02
Semen persicae / Amygdaluspersica L. / Seeds / -10.93
Semen trigonellae / Trigonellafoenum-graecum L. / Seeds / -9.84
Fructuscarpesii / Carpesiumabrotanoides L. / Fruit / -9.29
Radix gentianaemacrophyllae / Centianamacrophylla Pall. / Roots / -9.29
Rhizomaligustici / LigusticumsinenseOliv. / Rhizomes / -9.29
Radix curcumae / Curcuma wenyujin Y. H. Chen et C. Ling / Tubers / -8.74
Radix asteris / Aster tataricusL.f. / Rhizomes / -8.74
Folium pyrrosiae / Pyrrosia lingua (Thunb.) Farwell. / Leaves / -8.20
Herbaschizonepetae / SchizonepetatenuifoliaBriq. / Aerial parts / -7.65
Fructustribuli / Tribulusterrestris L. / Fruits / -7.65
Rhizomaacoritatarinowii / Acorustatarinowii Schott. / Rhizomes / -7.10
Pericarpiumcitrireticulataeviride / Citrus reticulataBlanco. / Pericarp / -7.10
Radix notoginseng / Panaxnotoginseng (Burk.) F. H. Chen. / Roots / -7.10
Rhizomasmilacisglabrae / Smilax glabraRoxb. / Rhizomes / -6.56
Rhizomasparganii / SpaganiumstoloniferumBuch.-Ham. / Tubers / -6.01
Herbaecliptae / Ecliptaprostrata L. / Aerial parts / -6.01
Herbaagrimoniae / AgrimoniapilosaLedeb. / Aerial parts / -6.01
Herbalycopi / LycopuslucidusTurcz. var. hirtus Regel / Aerial parts / -5.46
Rhizomaalpiniaeofficinarum / AlpiniaofficinarumHance. / Rhizomes / -5.46
Herbamoslae / Moslachinensis Maxim. / Aerial parts / -4.92
Caulis fibraureae / Tripterygium wilfordii Hook. f. / Stems / -3.83
Radix platycodi / Platycodon grandiflorum (Jacq.) A.DC. / Roots / -3.83
Herbadianthi / Dianthus superbus L. / Aerial parts / -3.83
Fructuschaenomelis / Chaenomelesspeciosa（Sweet）Nakai. / Fruits / -3.83
Semen vaccariae / Vaccariasegetalis (Neck.) Garcke. / Seeds / -3.83
Cacumenplatycladi / Platycladus orientalis (Zhou et al.) Franco. / Leaves / -2.19
Radix cynanchiatrati / CynanchumatratumBge. / Rhizomes / -2.19
Rhizomaanemarrhenae / AnemarrhenaasphodeloidesBge. / Rhizomes / -2.19
Floschrysanthemi / Chrysanthemum morifolium Ramat. / Inflorescences / -1.64
Flosalbiziae / AlbiziajulibrissinDurazz. / Inflorescences / -1.09
Semen trichosanthis / Trichosantheskirilowii Maxim. / Seeds / -0.55
Semen alliituberosi / Allium tuberosumRottl. / Seeds / 0.55
Herbaeupatorii / Eupatorium fortuneiTurcz. / Aerial parts / 2.73
Herbaasari / AsarumsieboldiiMiq. / Whole plants / 2.73
Radix scrophulariae / ScrophularianingpoensisHemsl. / Roots / 2.73
Radix ephedrae / Ephedra sinicaStapf. / Rhizomes / 3.28
Rhizomaalismatis / Alismaorientalis (Sam.) Juzep. / Tubers / 3.28
Fructuspiperislongi / Piper longum L. / Fruits / 3.28
Rhizomacurcumae / Curcuma phaeocaulis Val. / Rhizomes / 4.37
Rhizomacyperi / Cyperusrotundus L. / Rhizomes / 4.37
Radix adenophorae / Adenophoratetraphylla (Thunb.) Fisch. / Roots / 4.37
Herbacirsii / Cirsiumsetosum (Willd.) MB. / Aerial parts / 4.92
Radix bupleuri / Bupleurumchinense DC. / Roots / 5.46
Rhizomahomalomenae / Homalomenaocculta (Lour.) Schott. / Rhizomes / 6.01
Bulbusalliimacrostemonis / Allium macrostemonBge. / Bulbs / 6.56
Fructusfoeniculi / Foeniculum vulgare Mill. / Fruits / 7.10
Pericarpiumzanthoxyli / Zanthoxylumbungeanum Maxim. / Seeds / 7.65
Ramuluscinnamomi / Cinnamomum cassiaPresl / Bark / 7.65
Fructusschisandrae / Schisandrachinensis (Turcz.) Baill. / Fruits / 7.65
Fructustsaoko / AmomumtsaokoCrevost et Lemaire. / Fruits / 7.65
Radix angelicaedahuricae / Angelica dahurica (Fisch. ex Hoffm.) Benth. / Roots / 9.84
Folium artemisiaeargyi / Artemisia argyiLevl. etVant. / Leaves / 10.38
Fructuskochiae / Kochia scoparia (Zhou et al.) Schrad. / Fruits / 11.48
Herbapogostemonis / Pogostemoncablin (Blance) Benth. / Aerial parts / 12.02
Radix pseudostellariae / Pseudostellariaheterophylla (Miq.) Pax ex Pax et hoffm. / Tubers / 12.57
Radix astragaliseuhedysari / Astragalusmembranaceus (Fisch.) Bge.var. mongolicus (Bge.) Hsiao. / Roots / 13.11
Cortex moutanradicis / PaeoniasuffruticosaAndr. / Velamen / 13.67
Rhizomacynanchistauntonii / Cynanchumstauntonii (Decne.) Schltr.ex Level. / Rhizomes / 14.75
Rhizomaatractylodismacrocephalae / Atractylodesmacrocephalakoidz. / Rhizomes / 15.30
Fructusrosaelaevigatae / Rosa laevigataMichx. / Fruit / 16.94
Fructusevodiae / Evodiarutaecarpa (Juss.) Benth. / Fruits / 17.49
Radix codonopsis / Codonopsispilosula (Franch.) Nannf. / Roots / 17.49
Radix pulsatillae / Pulsatillachinensis (Bge.) Regel. / Roots / 18.58
Caulis perillae / Perillafrutescens (Zhou et al.) Britt. / Stems / 21.86
Radix achyranthisbidentatae / Achyranthesbidentata Bl. / Roots / 21.86
Radix ophiopogonis / Ophiopogonjaponicus(Thunb.) Ker-Gawl. / Tubers / 23.50
Fructusamomirotundus / AmomumkravanhPirre. exGagnep. / Fruit / 24.59
Caulis clematis armanoii / Clematis armandiiFranch. / Stems / 24.59
Herbaartemisiaescopariae / Artemisia scopariaWaldst. et Kit. / Aerial parts / 30.05
Radix stellariae / Stellariadichotoma L. var. lanceolataBge. / Roots / 41.53
*negative values indicate decrease, while positive values indicate increase, in methane production as compared to that of the control
Table S2
PCR primers used in real-time quantitative PCR assay
Target species / Primer sequence / ReferenceTotal bacteria / 5’-CGGCAACGAGCGCAACCC-3’ / Denman and McSweeney 2006
5’-CCATTGTAGCACGTGTGTAGCC-3’
Fibrobacter succinogenes / 5’-GTTCGGAATTACTGGGCGTAAA-3’ / Denman and McSweeney 2006
5’-CGCCTGCCCCTGAACTATC-3’
Ruminococcus flavefaciens / 5’-CGAACGGAGATAATTTGAGTTTACTTAGG-3’ / Denman and McSweeney 2006
5’-CGGTCTCTGTATGTTATGAGGTATTACC-3’
Ruminococcus albus / 5’-CCCTAAAAGCAGTCTTAGTTCG-3’ / Koike and Kobayashi 2001
5’-CCTCCTTGCGGTTAGAACA
Fungi / 5’-GAGGAAGTAAAAGTCGTAACAAGGTTTC-3’ / Denman and McSweeney 2006
5’-CAAATTCACAAAGGGTAGGATGATT-3’
Protozoa / 5’-GCTTTCGWTGGTAGTGTATT-3’ / Sylvester et al. 2004
5’-CTTGCCCTCYAATCGTWCT-3’
Methanogen / 5’-TTCGGTGGATCDCARAGRGC-3’ / Denman et al. 2007
5’-GBARGTCGWAWCCGTAGAATCC-3’
Denman SE, McSweeney CS (2006) Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen. FEMS MicrobiolEcol 58(3):572-582
Denman SE, Tomkins NW, McSweeney CS (2007) Quantitation and diversity analysis of ruminal methanogenic populations in response to the antimethanogenic compound bromochloromethane. FEMS MicrobiolEcol 62(3):313-322
Koike S, Kobayashi Y (2001) Development and use of competitive PCR assays for the rumen cellulolytic bacteria: Fibrobactersuccinogenes, Ruminococcusalbus and Ruminococcusflavefaciens. FEMS Microbiol Lett 204(2):361-366
Sylvester JT, Karnati SKR, Yu ZT, Morrison M, Firkins JL (2004) Development of an assay to quantify rumen ciliate protozoal biomass in cows using real-time PCR. J Nutr 134(12):3378-3384
Table S3
Relative abundance of bacterial and archaeal genera* (or genus-equivalent taxa) in the invitro cultures receiving the extracts of Perillafrutescensseeds
PF in vitro culture, mg/mL / SEM / P value0 / 0.2 / 0.4 / PF / Linear / Quadratic
Actinobacteria
unclassified JdFBGBact / 2.75E-4a / 2.06E-5ab / 6.86E-6b / 6.50E-5 / 0.03 / 0.02 / 0.17
Bacteroidetes
unclassified [Paraprevotellaceae] / 9.55E-3a / 9.28E-3a / 5.57E-3b / 7.58E-4 / <0.01 / <0.01 / 0.10
unclassified [Acidimicrobineae] / 6.18E-5a / 0b / 0b / 1.35E-5 / 0.02 / 0.01 / 0.10
unclassified YRC22 / 9.03E-3a / 8.44E-3a / 3.55E-3b / 5.41E-4 / <0.01 / <0.01 / 0.01
unclassified [Prevotellaceae] / 1.44E-2a / 1.03E-2b / 5.46E-3c / 9.98E-4 / <0.01 / <0.01 / 0.78
unclassified BS11 / 6.99E-3a / 6.74E-3a / 2.67E-3b / 6.20E-4 / <0.01 / <0.01 / 0.03
Paludibacter / 5.70E-4a / 2.95E-4b / 1.30E-4b / 6.76E-5 / <0.01 / <0.01 / 0.52
unclassified VC21_Bac22 / 1.92E-4a / 6.18E-5ab / 0b / 4.01E-5 / 0.02 / <0.01 / 0.50
Luteimonas / 1.26E-3a / 9.95E-4ab / 3.43E-5b / 3.05E-4 / 0.05 / 0.02 / 0.37
unclassified Ekhidnaceae / 6.32E-4ab / 1.01E-3a / 1.17E-4b / 1.76E-4 / 0.02 / 0.07 / 0.02
unclassified Sphingobacteriaceae / 1.17E-4a / 6.86E-5ab / 6.88E-6b / 2.61E-5 / 0.05 / 0.02 / 0.84
Pedobacter / 1.44E-4a / 1.10E-4ab / 5.49E-5b / 1.87E-5 / 0.02 / <0.01 / 0.66
Chloroflexi
unclassified Caldilineaceae / 2.61E-4a / 1.17E-4b / 4.12E-5b / 3.06E-5 / <0.01 / <0.01 / 0.38
Fibrobacteres
Fibrobacter / 2.03E-2a / 1.25E-2b / 1.81E-3c / 9.75E-4 / <0.01 / <0.01 / 0.26
Firmicutes
unclassified Christensenellaceae / 1.58E-2a / 1.11E-2ab / 8.65E-3b / 1.35E-3 / 0.01 / <0.01 / 0.50
Clostridium / 9.24E-3a / 8.23E-3a / 4.49E-3b / 6.59E-4 / <0.01 / <0.01 / 0.13
unclassified Lachnospiraceae / 3.10E-2a / 2.58E-2ab / 2.18E-2b / 1.93E-3 / 0.03 / <0.01 / 0.82
Pseudobutyrivibrio / 1.72E-2b / 1.85E-2b / 2.87E-2a / 1.27E-3 / <0.01 / <0.01 / 0.02
Shuttleworthia / 2.40E-3b / 3.46E-3ab / 4.54E-3a / 3.64E-4 / <0.01 / <0.01 / 0.98
Ruminococcus / 1.60E-2a / 1.36E-2ab / 1.07E-2b / 1.25E-3 / 0.05 / 0.02 / 0.91
Anaerovibrio / 1.55E-3b / 2.09E-3b / 4.48E-3a / 2.23E-4 / <0.01 / <0.01 / <0.01
Selenomonas / 7.19E-3b / 7.52E-3b / 1.33E-2a / 7.46E-4 / <0.01 / <0.01 / 0.02
Proteobacteria
Roseomonas / 4.81E-4b / 6.66E-4ab / 1.17E-3a / 1.64E-4 / 0.04 / 0.02 / 0.44
Zymomonas / 3.43E-5a / 0b / 0b / 7.59E-6 / 0.02 / 0.01 / 0.10
unclassified Burkholderiaceae / 3.43E-5b / 1.37E-5b / 2.13E-4a / 4.11E-5 / 0.01 / 0.01 / 0.06
unclassified Desulfobacteraceae / 8.92E-5a / 3.43E-5b / 0b / 1.41E-5 / <0.01 / <0.01 / 0.57
unclassified Desulfobulbaceae / 1.69E-3ab / 1.98E-3a / 4.67E-4b / 3.77E-4 / 0.04 / 0.05 / 0.08
unclassified Succinivibrionaceae / 3.14E-3c / 5.26E-3b / 9.86E-3a / 3.02E-4 / <0.01 / <0.01 / <0.01
Ruminobacter / 1.64E-1b / 2.15E-1b / 3.58E-1a / 0.014 / <0.01 / <0.01 / 0.03
Succinivibrio / 3.30E-2c / 4.60E-2b / 7.68E-2a / 0.003 / <0.01 / <0.01 / 0.03
unclassified OM60 / 5.42E-4a / 5.01E-4ab / 7.55E-5b / 1.09E-4 / 0.03 / 0.01 / 0.18
unclassified Sinobacteraceae / 7.62E-4b / 6.93E-4b / 1.32E-3a / 1.39E-4 / 0.02 / 0.02 / 0.07
Spirochaetes
Sphaerochaeta / 1.66E-3a / 1.41E-3ab / 6.66E-4b / 2.32E-3 / 0.04 / 0.01 / 0.41
Tenericutes
unclassified Anaeroplasmataceae / 8.38E-4a / 6.11E-4a / 2.06E-4b / 7.80E-5 / <0.01 / <0.01 / 0.37
Anaeroplasma / 1.81E-3a / 1.48E-3ab / 9.61E-4b / 1.57E-4 / 0.01 / <0.01 / 0.65
unclassified RFN20 / 9.47E-4a / 5.70E-4b / 6.18E-4b / 8.00E-5 / 0.02 / 0.02 / 0.06
Verrucomicrobia
unclassified R4-41B / 1.65E-4b / 1.65E-4b / 4.05E-4a / 5.00E-5 / 0.01 / <0.01 / 0.08
Euryarchaeota
Methanobrevibacter / 8.30E-1b / 8.44E-1ab / 8.59E-1a / 6.68E-3 / 0.04 / 0.02 / 0.94
M. ruminantium / 1.26E-1 / 1.24E-1 / 1.31E-1 / 2.19E-3 / 0.13 / 0.14 / 0.14
M.millerae / 5.64E-3a / 4.84E-3a / 3.28E-3b / 2.92E-4 / <0.01 / <0.01 / 0.32
unclassified Methanobrevibacter / 6.98E-1b / 7.14E-1ab / 7.24E-1a / 5.20E-3 / 0.02 / <0.01 / 0.61
unclassified Methanobacteriaceae / 4.27E-2a / 3.16E-2b / 2.37E-2c / 1.00E-3 / <0.01 / <0.01 / 0.23
Methanosphaera / 8.40E-3b / 1.09E-2b / 1.52E-2a / 6.83E-4 / <0.01 / <0.01 / 0.34
* Only the genera whose relative abundance was significantly affected are presented
a / bFig.S1Rarefaction curves based on operational taxonomic units (OTUs) clustered at 3% divergence for the bacterial community (a) and the archaeal community (b) of each sample
Correspondence:
Tel: +86 571 88982389. Fax: +86 571 88982930. E-mail: (J.K. Wang).Post address: College of Animal Sciences, Zhejiang University, Yuhangtang Road 866#, Hangzhou, Zhejiang, China.