Journal of Medicine and Health (JMH) Vol. 6 No. 1 February 2024 e-ISSN: 2442-5257 https://doi.org/10.28932/jmh.v6i1.7689 Research Article Prebiotic Effect of Cogon Grass Root Extract on Stimulating Lactic Acid Bacteria Growth in Mice Intestine Efek Prebiotik Ekstrak Akar Alang-alang dalam Menstimulasi Pertumbuhan Bakteri Asam Laktat pada Usus Halus Mencit Gita W Pradini1^, Nisa Fauziah1^, Shinta Asarina2, Neni Anggraeni3, Rini Widyastuti4, Mas Rizky AA Syamsunarno1* 1Department of Biomedical Science, Faculty of Medicine, Universitas Padjadjaran Jl. Raya Jatinangor-Sumedang Km 21 Jatinangor, Sumedang 2Microbiology Laboratory, Faculty of Medicine, Universitas Padjadjaran Jl. Eijkman No. 38 Bandung 3 Doctoral program in medical science, graduate school of comprehensive human science, University of Tsukuba 1 Chome-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Jepang 4 Department of Veterinary Product, Faculty of Veterinary, Universitas Padjadjaran Jl. Raya Jatinangor-Sumedang Km 21 Jatinangor, Sumedang ^These authors have contributed equally to this work. *Corresponding author Email: rizky@unpad.ac.id Received: October 31, 2023 Accepted: February 15, 2024 Abstract Lactic acid bacteria (LAB) are known for their numerous beneficial effects for health and their colonization within intestines is affected by prebiotic consumption. Cogon grass root is an herbal medicine which contain substantial quantities of polyphenols. Recently, many studies suggested polyphenols as a novel group of prebiotics. Therefore, this study aimed to explore prebiotic effect of cogon grass root extract (CGRE) on stimulating growth of LAB in mice intestine. Mice (n =7/group) were divided into three groups; group A (control), group B and C received CGRE orally with daily dosage 90 and 115mg/kg BW, respectively. After 14 days of treatment, all mice were sacrificed. LAB was isolated and counted from ileum. Additional biochemical tests were performed to confirm the bacteria as LAB. Further identification of representatives LAB was performed with API-CHL-50 System. We found increased growth and diversity of LAB in the treatment group with optimum growth stimulation at CGRE dosage 90mg/KgBW daily (group B). Further identification showed Lactococcus lactis was the most frequent LAB found in all groups, with additional Lactobacillus paracasei and Lactobacillus curvatus were found in the treatment group. These findings support additional benefit of CGRE as prebiotics by stimulating LAB growth and diversity in mice intestine. Keywords: cogon grass root; lactic acid bacteria; polyphenols; prebiotic How to Cite: Pradini GW, Fauziah N, Asarina S, Anggraeni N, Widyastuti R, Syamsunarno MRAA. Prebiotic Effect of Cogon Grass Root Extract on Stimulating Lactic Acid Bacteria Growth in Mice Intestine. Journal of Medicine and Health. 2024; 6(1): 34-44. DOI: https://doi.org/10.28932/jmh.v6i1.7689 J Med Health.2024;6(1):34-44 34 Journal of Medicine and Health Vol. 6 No. 1 February 2024 Prebiotic Effect of Cogon… e-ISSN: 2442-5257 Research Article © 2023 The Authors. This work is licensed under a Creative Commons AttributionNonCommercial 4.0 International License. Abstrak Bakteri asam laktat (BAL) diketahui memiliki banyak manfaat untuk kesehatan dan kolonisasinya di usus halus dipengaruhi oleh konsumsi prebiotik. Akar alang-alang adalah salah satu obat herbal yang memiliki polifenol dengan jumlah yang bermakna. Banyak studi terkini yang merekomendasikan polifenol sebagai kelompok baru prebiotik. Penelitian ini bertujuan untuk mengetahui efek prebiotik dari akar alang-alang dalam menstimulasi pertumbuhan BAL dalam usus halus mencit. Mencit (n=7/kelompok) dibagi menjadi tiga kelompok: kelompok A (kontrol), kelompok B dan kelompok C (uji) yang masing-masing mendapatkan ekstrak akar alang-alang secara oral dengan dosis 90 dan 115 mg/KgBB setiap harinya. Setelah perlakukan selama 14 hari, semua mencit dikorbankan. BAL diisolasi dan dihitung koloninya dari ileum. Uji biokimiawi dilakukan untuk mengkonfirmasi bakteri yang diisolasi sebagai BAL. Identifikasi lebih lanjut dengan menggunakan API-CHL-50 system dilakukan untuk beberapa isolat yang representatif. Hasil penelitian menunjukkan peningkatan jumlah koloni dan diversitas BAL pada kelompok uji dengan dosis optimal 90mg/KgBB per hari (kelompok B). Identifikasi spesies BAL menunjukkan Lactococcus lactis merupakan BAL yang tersering ditemukan di seluruh kelompok. Lactobacillus paracasei dan Lactobacillus curvatus ditemukan hanya pada kelompok uji. Penelitian ini menunjukkan manfaat lain dari ekstrak alang-alang sebagai prebiotik dengan menstimulasi pertumbuhan dan diversitas BAL dalam usus halus mencit. Kata kunci: akar alang-alang; bakteri asam laktat; polifenol; prebiotik Introduction Gastrointestinal microbiota is known to have a significant contribution to both health and disease in humans. The balance of gastrointestinal microbiota diversity is very important for intestinal homeostasis, and it has an impact on human health and longevity. 1-3 Microbiota of the gastrointestinal tract are diverse, it include the beneficial lactic acid bacteria (LAB) which contribute significantly to maintain intestinal homeostasis.4 LAB is known to have beneficial effect for improving gastrointestinal integrity, prevent gastrointestinal infection, relieving constipation, reduce inflammatory conditions, and promote general wellness. 5-6 LAB mostly inhabit jejunum and ileum, with Lactobacilli and Streptococci as dominant genera.7,8 The LAB is colonized and attached to the mucus layer of the intestines and acts as an immunomodulator. 7 The colonization and abundance of these bacteria are affected by dietary patterns, particularly by prebiotic consumption.8,9 During a prebiotic deficient diet, it was revealed that the condition lead to development of hypertensinogenic gut microbiota, thus increased risk for hypertension and its complications in the future.10 Recently, evidence suggested polyphenols can act as prebiotics. 11–13 Without being absorbed in the small intestines, their presence in the lumen of the gastrointestinal tract can modulate gastrointestinal microbiota.12,14,15 Interestingly, dietary polyphenols can act differently J Med Health.2024;6(1):34-44 35 Journal of Medicine and Health Vol. 6 No. 1 February 2024 Prebiotic Effect of Cogon… e-ISSN: 2442-5257 Research Article on LAB and pathogenic bacteria, it enhances LAB adhesion on intestinal epithelial cells but reduces the adhesion of pathogenic bacteria.12,13,16The main sources of polyphenols in humans are derived from plants such as fruit, vegetables, herbs, cocoa and beverages (e.g. tea, coffee, and wine).9,13,17 Other sources for polyphenols are traditional herbal medicines.12,18–20 The root of Cogon grass (Imperata cylindrica (L.)), is a common traditional medicine in Indonesia,21 has been proven to have antioxidant and anti-inflammatory properties due to its polyphenols contents.22–25 Previous study found pretreatment of mice with oral cogon grass root extract (CGRE) for two weeks, significantly reduced serum cholesterol levels after acute olive oil loading.25 There is evidence polysaccharide from root of I. cylindrica can reduce cholesterol level,26 and we want to know if it also has impact on LAB. Thefore, the objective of this study is to explore whether the GCRE has prebiotic effects to stimulate LAB growth in mice intestine. Methods Plant Material and Extract CGRE preparation was conducted as previously described.26 Briefly, authenticated cogon grass roots (Imperata cylindrica (L.) Beauv.) from local region of Java was dried and powdered. The grass root powder was macerated by 96% ethanol for 72 hours to extract its active compounds. The macerated pulp then filtered and evaporated to remove ethanol excess. The extract then diluted with 0.5% carboxyl methyl cellulose (CMC). Animal Study The study was approved by the Ethics Review Committee Faculty of Medicine Universitas Padjadjaran Number 1263/UN6.C10/PN/2017. Adult male DDY-mice with aged 812 weeks were obtained from PT. Bio Farma (Bandung, Indonesia). Mice were housed in a room with 12/12 h light and dark cycle with good air circulation. Food and drinking water were provided with no restriction. The number animal tested for each group was determined using formula of minimum and maximum sample size for group comparison one-way ANOVA. The formula consist of minimum number of animals per group: n = (10/k) + 1 and maximum number of animals per group: n = (20/k) + 1. The “k” represents number of treatment group.27 Based on the formula, we include seven mice for each group. Mice were divided into three groups, which are: group A as control, group B and group C as treatment group which received CGRE orally with daily dosage 90 and 115mg/kg BW in 200 μl of 0.5 % CMC, respectively. CGRE was given once a day orally for 14 days. At the end of the experiment, mice J Med Health.2024;6(1):34-44 36 Journal of Medicine and Health Vol. 6 No. 1 February 2024 Prebiotic Effect of Cogon… e-ISSN: 2442-5257 Research Article were euthanized, and ileum was aseptically isolated (±3.5 cm) from each mouse and placed into a sterile tube containing 2 ml of sterile NaCl 0.85%. Sample were immediately stored in the -40oC freezer until it ready to perform LAB isolation and counting.28 Isolation of Lactic Acid Bacteria (LAB) MRS (de Man, Ragosa, and Sharpe) agar plates with the addition of CaCO3 1% (MRS+CaCO31%) were used for LAB isolation. CaCO3 1% was added to distinguish acidproducing bacteria, was identified by the presence of a clear zone surrounding the suspected colony. Stored ileum samples were thawed and aseptically cut and minced using sterile scissors and homogenized using a vortex. Homogenized samples then serially diluted from 10-1 to 10-4 fold in sterilized water. One hundred microliters from each serial dilution were spread onto the surface of MRS+CaCO31% agar plates and incubated at 37 oC under the microaerophilic condition (5% of CO2) for 48 hours. Colonies of acid-producing bacteria were identified by the presence of a clear zone around each colony. The number of LAB colonies from each sample was calculated by using total plate count method. Five colonies from each intestine sample were selected randomly from plates containing between 30-300 colonies, each colony was Gram stained to ensure the purity of the Gram-positive colony. Each of colony were sub-cultured and incubated at 37oC for 48 hours under microaerophilic conditions. Purified LAB colonies were stored at -80oC in MRS broth with 10% glycerol (v/v) until it ready for further identification of LAB characteristics. Morphological, Biochemical and Physiological Test of Probiotics LAB The purified LAB colony was grown on MRS+CaCO3 1% agar for 48 hours at 37 oC. Morphology, gram staining response, biochemical and physiological test were observed afterward. Catalase activity was confirmed by placing a loopful of the LAB colony on the glass slide and mixed with a drop of 3% H 2O2. LAB was identified for its negative catalase activity. Gas production from glucose fermentation was observed by incubating a loopful of LAB colony into 5 ml phenol red glucose broth (glucose 1%, peptone 1%, NaCl 0.5%, and phenol red 0.4%) in Durham tube test for 48 hours at 37 oC. Gas production indicated for heterofermentative LAB, whereas homofermentative LAB did not produce gas.29 . Physiological characterization of probiotics LAB (acid and salt resistance) was observed with growth at pH 3.0, 3.5, 4.0, 4.5 and 7.0 in MRS broth after incubation at 37°C for 2 and 4 days. Salt resistance of probiotics LAB was tested in MRS broth containing 3.0 and 6.5% NaCl at 37 °C for 2 and 4 days.30 All incubation for LAB growth was performed under microaerophilic condition. J Med Health.2024;6(1):34-44 37 Journal of Medicine and Health Vol. 6 No. 1 February 2024 Prebiotic Effect of Cogon… e-ISSN: 2442-5257 Research Article Lactic Acid Production The purified colony of LAB was grown in MRS broth for 24 hours at 37 oC under microaerophilic condition. Approximately 10% v/v of each purified colony was inoculated into 40 ml glucose broth 37oC. After 48 hours of fermentation, lactic acid production was quantified with acid-base titration method. Two replicates were made for each treatment. Confirmation of Lactic Acid Bacteria Species Carbohydrates fermentation of twenty LAB isolates were determined using API 50 CHL in conjunction with API 50 CHL medium (bioMérieux, France) for identification of LAB with L. plantarum as control. The APILAB PLUS database identification software was used to interpret the result. Statistical Analysis. Statistical analysis using ANOVA was performed to find significant differences in LAB count between control, 90 mg dose and 115 mg dose of CGRE treatment. A significance level of p < 0.05 was used. Results Lactic Acid Bacteria (LAB) Growth Based on LAB growth on MRS+CaCO3 1% agar, sample group A (control) had mean colony counting of 210(±247) x 103 cfu/ml, group B (dosage 90 mg/KgBW) had mean colony counting of 5,811(±5595) x 103 cfu/ml and group C (dose 115 mg/KgBW) had mean colony counting 1,871 (±2038) x 103 cfu/ml (Figure 1). From this result, the optimum growth stimulation for LAB in mice intestines is after supplementation with CGRE with daily dosage 90mg/KgBW for 14 days. Morphological, biochemical and physiological properties of LAB isolated from mice intestines A total of 25, 27 and 33 LAB isolates (total n=85) were collected from mice groups A, B, and C respectively. Further LAB characterization was done as in table 1. All presumptive LAB isolates were Gram-positive and catalase-negative. To confirm the isolates as a lactic acid producer, lactic acid production was measured resulting in mean lactic acid concentration from all group ranged between 9.4 mM–10.4 mM. Salt and acid tolerability were measured to J Med Health.2024;6(1):34-44 38 Journal of Medicine and Health Vol. 6 No. 1 February 2024 Prebiotic Effect of Cogon… e-ISSN: 2442-5257 Research Article characterized functional properties of isolated bacteria, the result showed most isolates were acid tolerable at pH 4.0 and salt tolerable for up to 4 days. Sugar fermentation types from all isolates were homofermentative (no gas produced from glucose fermentation).29 Confirmation of LAB Species To confirm the species of LAB isolated from mice intestines, 16 representative samples from the control group (n=9) and treatment group (group B, n=7) were randomly selected to further identified with API CHL 50 system (table 2). Figure 1 The LAB Colony Counting Legend: A (control), B (CGRE dosage 90 mg/KgBW), C (CGRE dosage 100 mg/KgBW). Asterix (*) indicate p<0.05 (statistically significant different between 2 groups: A vs B and A vs C) Table 1 Characterization of Isolated LAB Characteristics Group A Group B Group C 27 33 All positive All negative Homofermentative All negative 25 12% Cocobacilli 8% Bacilli 80% Cocci All positive All negative Homofermentative All negative All positive All negative Homofermentative All negative 10.4 ± 1.1 mM 10.1 ± 2.7 mM 9.4 ± 1.1 mM Number of Isolates Morphology Gram stain Catalase activity Fermentation type Gas production Lactic acid concentration (mean±SD) Growth in NaCl 3% 6.5% Growth at pH 3.0 3.5 4.0 J Med Health.2024;6(1):34-44 100% Cocci 94% Cocci 6% Bacilli Day 2 100% Day 4 100% Day 2 96% Day 4 96% Day 2 100% Day 4 100% 100% 100% 96% 96% 100% 100% Day 2 40.7% 88.8% 100% Day 4 0 25.9% 100% Day 2 28% 60% 92% Day 4 0 0 88% Day 2 9% 75.6% 97% Day 4 0 6% 94% 39 Journal of Medicine and Health Vol. 6 No. 1 February 2024 Prebiotic Effect of Cogon… e-ISSN: 2442-5257 Research Article 4.5 7.0 100% 100% 100% 100% 96% 100% 88% 100% 100% 100% 97% 100% Discussion Marked changes on gastrointestinal microbiota diversity or dysbiosis have linked to major human diseases, including autoimmune and inflammatory diseases (obesity, hypertension, cardiovascular disease, and type 2 diabetes mellitus).2-3,31 Modulation of gastrointestinal microbiota to a healthy state could ease many health problems, and it can be achieved by consumption of prebiotics.32 Herbal medicine could exert its benefit through its polyphenols compound by modulating gastrointestinal microbiota. Its polyphenols not only support the growth of beneficial bacteria, but also suppress pathogenic bacterial growth within intestine.15,33 Our study found administration of herbal medicine CGRE, with daily dosage of 90mg/KgBW, intraorally for 14 days significantly increase the growth of LAB in mice intestine (figure 1), suggesting its effect as prebiotics. Therefore, supporting application of CGRE as an alternative for dietary source of prebiotics and might be beneficial to alleviate gastrointestinal problems caused by prebiotics defiency. LAB has been proved to have beneficial effects on human health through metabolism regulation, infection control, and inflammation/allergy modulation.34,35 According to phenotypic characteristics data, we found more diverse LAB morphology in groups who received CGRE than the control group (table 1). Representative samples were chosen from treatment group (B) and control (A) to confirm the species of LAB. All representative samples which are found from the control group were identified as Lactococcus lactis ssp lactis 1 (n=9), while from the treatment group (B) Lactobacillus curvatus (n=1), Lactocobacillus paracasei ssp. paracasei 3 (n=4), and Lactococcus lactis ssp lactis 1 (n=2) were found. This finding represents criteria for defining a prebiotic, which is the ability to stimulate selectively the growth and/or activity of intestinal bacteria associated with health and wellbeing.8 Similar evidence was also found from a study in metabolic syndrome patients who consumed polyphenol-rich red wine and presenting a higher number of intestinal barrier protector bacteria (Bifidobacterium and Lactobacillus genera) and butyrate-producing bacteria (Faecalibacterium prausnitzii and Roseburia) while the number of E. coli and E. cloacae were decreasing.33 Table 2 Species Identification of Representatives LAB Samples Group Control (Group A) Treatment (Group B) J Med Health.2024;6(1):34-44 Species Lactococcus lactis ssp lactis 1 Lactobacillus curvatus Lactococcus lactis ssp lactis 1 Lactocobacillus paracasei ssp paracasei 3 Isolates (n) 9 1 2 4 40 Journal of Medicine and Health Vol. 6 No. 1 February 2024 Prebiotic Effect of Cogon… e-ISSN: 2442-5257 Research Article J Med Health.2024;6(1):34-44 41 Journal of Medicine and Health Vol. 6 No. 1 February 2024 Prebiotic Effect of Cogon… e-ISSN: 2442-5257 Research Article These findings suggest an effect of polyphenols to promote growth and diversity of beneficial bacteria, including LAB. Increased diversity of LAB within the treatment group might also be stimulated with cross-feeding phenomena, which is the utilization of prebiotics byproducts (e.g. exopolysaccharides, short-chain fatty acids) produced by certain microbes as substrates for growth of other microbes within the same community.32 LAB can synthesize bioactive molecules, including polysaccharides, such as exopolysaccharides.35 Biosynthesis of polysaccharides is increased with the presence of polyphenols which might provide the metabolic source for another sub-dominant beneficial LAB genera within intestines.33 Thus, increasing the amount and diversity of the beneficial LAB. Although much evidence shows positive stimulation of polyphenols on growth and functional properties of LAB,16,33,36other studies also found inhibition of polyphenols on certain LAB growth which usually depend on LAB strain, concentration and type of polyphenols given.39 Our study found a higher dose of CGRE (115 mg/kg BW daily) reduced the LAB colony within the intestine. This finding confirmed polyphenol concentration-dependent on LAB growth stimulation. This study is limited by CGRE used in this study which is crude extract and it had not been characterized for specific polyphenols content. Nevertheless, another study found twelve phenolic compounds present in the cogon-grass root that belongs to the group of flavonoids, simple phenols, phenolic acids, coumarins, and lignans.38 Moreover, the impact of CGRE on LAB growth might be affected by polysaccharides that naturally present in cogon-grass roots.39 Polysaccharides, particularly with prebiotic properties, can modify intestinal microbiota with growth stimulation of beneficial microbiota, including lactic acid bacteria.40 Conclusion This study revealed another benefit of CGRE, a traditional herbal medicine, as prebiotics. The prebiotic effects showed by CGRE stimulation on LAB growth and diversity in mice intestines with optimal daily dosage at 90 mg/kg BW for 14 days. Our findings complete the knowledge of CGRE’s beneficial effect and its mechanism, particularly in modulating gut microbiota through the stimulation of LAB growth. Funding This study was conducted under a grant from the Indonesian Ministry of Research and Higher Education year 2017. J Med Health.2024;6(1):34-44 42 Journal of Medicine and Health Vol. 6 No. 1 February 2024 Prebiotic Effect of Cogon… e-ISSN: 2442-5257 Research Article References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. Olvera-Rosales LB, Cruz-Guerrero AE, Ramírez-Moreno E, Quintero-Lira A, Contreras-López E, Jaimez-Ordaz J, et al. Impact of the Gut Microbiota Balance on the Health-disease Relationship: The Importance of Consuming Probiotics and Prebiotics. Foods. 2021;10(6):1261. Ding RX, Goh WR, Wu RN, Yue XQ, Luo X, Khine WWT, et al. Revisit Gut Microbiota and Its Impact on Human Health and Disease. JFDA. 2019;27(3):623-31. Valdes AM, Walter J, Segal E, Spector TD. Role of the gut microbiota in nutrition and health. BMJ. 2018;361:3644. Lu S. The role of Lactic Acid Bacteria in Gut Microbiota and Mucosal Immune System. South Fla. 2021;2(3): 3918-25. Qin D, Ma Y, Wang Y, Hou X, Yu L. Contribution of Lactobacilli on Intestinal Mucosal Barrier, and Diseases: Perspectives and Challenges of Lactobacillus casei. Life.2022;12(11):1910. Dempsey E, Corr SC. Lactobacillus spp. for Gastrointestinal Health: Current and Future Perspectives. Front. Immunol. 2022;6(13):840245. Donaldson GP, Lee SM, Mazmanian SK. Gut biogeography of the bacterial microbiota. Nat. Rev. Microbiol. 2015;14(1):20–32. Nishiyama K, Sugiyama M, Mukai T. Adhesion properties of lactic acid bacteria on intestinal mucin. Microorganisms. 2016;4(3). Wang S, Xiao Y, Tian F, Zhao J, Zhang H, Zhai Q, et al. Rational use of prebiotics for gut microbiota alterations: Specific bacterial phylotypes and related mechanisms. J. Funct. Foods. 2020; 66:103838. Kaye DM, Shihata WA, Jama HA, Tsyganov K, Ziemann M, Kiriazis H, et al. Deficiency of Prebiotic Fiber and Insufficient Signaling Through Gut Metabolite-Sensing Receptors Leads to Cardiovascular Disease. Circulation. 2020;141(17):1393-403. Pickard JM, Zeng MY, Caruso R, Núñez G. Gut microbiota: Role in pathogen colonization, immune responses, and inflammatory disease. Immunol. Rev. 2017;279(1):70–89. Rodríguez-Daza MC, Pulido-Mateos EC, Lupien-Meilleur J, Guyonnet D., Desjardins Y, Roy D. PolyphenolMediated Gut Microbiota Modulation: Toward Prebiotics and Further. Front. Nutr. 2021;28(8):689456. Gwiazdowska D, Juś K, Jasnowska-Małecka J, Kluczyńska K. The Impact of Polyphenols on Bifidobacterium growth. Acta Biochim Pol. 2015;62(4):895–901 Plamada D, Vodnar DC. Polyphenols-gut microbiota interrelationship: A transition to a new generation of prebiotics. Nutrients. 2022;14(1):137. Milutinović M, Dimitrijević-Branković S, Rajilić-Stojanović M. Plant Extracts Rich in Polyphenols as Potent Modulators in the Growth of Probiotic and Pathogenic Intestinal Microorganisms. Front Nutr. 2021;30(8):688843. de Souza EL, de Albuquerque TMR, dos Santos AS, Massa NML, de Brito Alves JL. Potential interactions among phenolic compounds and probiotics for mutual boosting of their health-promoting properties and food functionalities-A review. Crit. Rev. Food Sci Nutr. 2019;59(10):1645–59. Abbas M, Saeed F, Anjum FM, Afzaal M, Tufail T, Bashir MS, et al. Natural polyphenols: An overview. Int. J Food Prop. 2017;20(8):1689–99. Hano C, Tungmunnithum D. Plant Polyphenols, more than Just Simple Natural Antioxidants: Oxidative Stress, Aging and Age-Related Diseases. Medicines. 2020;7(5):26 Klinder A, Shen Q, Heppel S, Lovegrove JA, Rowland I, Tuohy KM. Impact of increasing fruit and vegetables and flavonoid intake on the human gut microbiota. Food Funct. 2016;7(4):1788–96. Singh AK, Cabral C, Kumar R, Ganguly R, Rana HK, Gupta A, et al. Beneficial effects of dietary polyphenols on gut microbiota and strategies to improve delivery efficiency. Nutrients. 2019;11(9):2216. Susanto AH, Dwiati M. Assessment of cogon grass (Imperata cylindrica (L.) P. beauv.) genetic variation in Java, Indonesia using atpB-rbcL and trnL-F intergenic spacer. Biodiversitas. 2022;23(5):2760-7. Sytar O, Hemmerich I, Zivcak M, Rauh C, Brestic M. Comparative analysis of bioactive phenolic compounds composition from 26 medicinal plants. Saudi J. Biol. Sci. 2018;25(4):631–41. Tungmunnithum D, Thongboonyou A, Pholboon A, Yangsabai A. Flavonoids and Other Phenolic Compounds from Medicinal Plants for Pharmaceutical and Medical Aspects: An Overview. Medicines. 2018;5(3):93. Hidayat S, Rachmadiyanto AN. Utilization of Alang-Alang (Imperata cylindrica (L.) Raeusch.) as Traditional Medicine in Indonesian Archipelago. Proceedings The 1st SATREPS Conference. 2017; 1:82-89. Indriyanti RA, Ariyanto EF, Usman HA, Effendy RR, Dhianawaty D. Quantification of total polyphenols and flavonoids, antioxidant activity, and Sinensetin and Imperatorin contents of Imperata cylindrica root ethanol extract. Pharmacogn J. 2022;14(4):327–37. Anggraeni N, Syamsunarno MRAA, Mukarromah GR, Zada A, Triatin RD, Pamela Y, et al. Low Serum Cholesterol in Mice Pre-treated with Imperata cylindrica (L.) after Acute Olive Oil Gavage. KnE Life Sci. 2017;3(6):460. Arifin WN, Zahiruddin WM. Sample size calculation in animal studies using resource equation approach. Malays J Med Sci. 2017;24(5):101–5. Bayu HH, Isolation and Identification of Lactic Acid Bacteria from Channa sp. as Potential Probiotic. Jurnal Pembelajaran dan Biologi Nukleus. 2023;9(1):75-84. J Med Health.2024;6(1):34-44 43 Journal of Medicine and Health Vol. 6 No. 1 February 2024 Prebiotic Effect of Cogon… e-ISSN: 2442-5257 Research Article 29. Mejia-Gomez CE, Balcázar N., Isolation, characterisation and continuous culture of Lactobacillus spp. and its potential use for lactic acid production from whey. Food Sci. Technol. 2020;40(4):1-8. 30. Ni K, Wang Y, Li D, Cai Y, Pang H. Characterization, identification, and application of lactic acid bacteria isolated from forage paddy rice silage. PLoS One. 2015;10(3):e0121967. 31. Elias-Oliveira J, Leite JA, Pereira ÍS, Guimarães JB, Manso GM da C, Silva JS, et al. NLR and Intestinal Dysbiosis-Associated Inflammatory Illness: Drivers or Dampers? Front Immunol. 2020;11(11):1810. 32. Holscher HD. Dietary fiber and prebiotics and the gastrointestinal microbiota. Gut Microbes. 2017;8(2):172–84 33. Moreno-Indias I, Sánchez-Alcoholado L, Pérez-Martínez P, Andrés-Lacueva C, Cardona F, Tinahones F, et al. Red wine polyphenols modulate fecal microbiota and reduce markers of the metabolic syndrome in obese patients. Food Funct. 2016;7(4):1775–87 34. Moorthy M, Sundralingam U, Palanisamy UD. Polyphenols as prebiotics in the management of high-fat dietinduced obesity: A systematic review of animal studies. Foods. 2021;10:299 35. Hakim BNA, Xuan NJ, Oslan SNH. A Comprehensive Review of Bioactive Compounds from Lactic Acid Bacteria: Potential Functions as Functional Food in Dietetics and the Food Industry. Foods. 2023;12(15):2850. 36. Chan CL, Gan RY, Shah NP, Corke H. Polyphenols from selected dietary spices and medicinal herbs differentially affect common food-borne pathogenic bacteria and lactic acid bacteria. Food Control. 2018;92:437–43. 37. dos Santos AS, de Albuquerque TMR, de Brito Alves JL, de Souza EL. Effects of Quercetin and Resveratrol on in vitro Properties Related to the Functionality of Potentially Probiotic Lactobacillus Strains. Front. Microbiol. 2019;10:2229. 38. Nayim P., Mbaveng AT., Sudhir K., Wamba BEN., Sanjukta M., et al. Phytochemistry and pharmacology of Imperata cylindrica: A comprehensive review. Invest Med Chem Pharmacol.2023;6(1):76 39. Rosnizar R., Muliani F., Ramli IM., Eriani K. The Immunostimulant Effects of Alang-Alang (Imperata cylindrica) Roots Extract on BALB/c Male Mice (Mus musculus). Adv. Biol. Sci. Res. 2022;22 40. Álvarez-Mercado AI., Plaza-Diaz J. Dietary Polysaccharides as Modulators of the Gut Microbiota Ecosystem: An Update on Their Impact on Health. Nutrients 2022;14(19):4116 J Med Health.2024;6(1):34-44 44