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Planta Med 2017; 83(01/02): 87-96
DOI: 10.1055/s-0042-110097
Biological and Pharmacological Activity
Original Papers
Georg Thieme Verlag KG Stuttgart · New York

In Vitro Chondroprotective Potential of Extracts Obtained from Various Phyllantus Species

Kittisak Buddhachat
1   Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand
,
Siriwadee Chomdej
1   Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand
,
Waranee Pradit
2   Science and Technology Research Institute, Chiang Mai University, Chiang Mai, Thailand
,
Korakot Nganvongpanit
3   Department of Veterinary Biosciences and Public Health, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai, Thailand
,
Siriwan Ongchai
4   Thailand Excellence Center for Tissue Engineering and Stem Cells, Department of Biochemistry and the Center of Excellence for Innovation in Chemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
› Author Affiliations
Further Information

Publication History

received 02 December 2015
revised 13 May 2016

accepted 29 May 2016

Publication Date:
24 June 2016 (online)

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Abstract

Phyllanthus amarus has been proven to exhibit chondroprotection. Regarding the morphological similarities among Phyllanthus species, we were attracted to evaluate the chondroprotective potential of Phyllanthus species including P. amarus obtained from Chiang Mai and Phuket, Phyllanthus urinaria L., Phyllanthus urinaria subsp. chamaepeuce, Phyllanthus debilis, and Phyllanthus airy-shawii using interleukin-1β-induced degradation of cartilage explants. The ethanolic extracts of the plants were evaluated for major lignans, phyllanthin, and hypophyllanthin by HPLC and further measurements of the total contents of flavonoids and phenolic compounds along with the assays for antioxidant and anti-collagenase activities. The interleukin-1β-induced cartilage explant degradation was performed with/without the extracts at concentrations of 50–250 µg/mL. After 4–14 days of incubation, the medium was assayed for the level of sulfated glycosaminoglycans while the explants were measured for the remaining content of uronic acid. Proteoglycan intensity in the explants was determined by safranin O staining. Diacerein, the antiarthritic agent, was used as the positive control. Although the two major lignans were found in P. amarus from Chiang Mai, P. amarus from Phuket, and P. urinaria L. extracts, similar chondroprotective activities were observed in all Phyllanthus extracts. Total phenolic content and total flavonoid content of the extracts showed a correlation with antioxidation, whereas the total phenolic content correlated with anti-collagenase activity. Among the six extracts, P. airy-shawii showed the greatest antioxidant and collagenase inhibitory activities. The results revealed that chondroprotective activities of all of the extracts of Phyllanthus species might result from an additive or synergistic influence of some constituents of these plants, which could be considered for antiarthritic purposes.

Key words

Phyllanthus - Phyllanthaceae - antioxidation - anti-collagenase - osteoarthritis - proteoglycan
 

  • References

  • 1 Felson DT. An update on the pathogenesis and epidemiology of osteoarthritis. Radiol Clin North Am 2004; 42: 1-9
    CrossrefPubMedGoogle Scholar
  • 2 Goldring MB. Update on the biology of the chondrocyte and new approaches to treating cartilage diseases. Best Pract Res Clin Rheumatol 2006; 20: 1003-1025
    CrossrefPubMedGoogle Scholar
  • 3 Mahajan A, Verma S, Tandon V. Osteoarthritis. J Assoc Physicians India 2005; 53: 634-641
    PubMedGoogle Scholar
  • 4 Graham DJ. COX-2 inhibitors, other NSAIDs, and cardiovascular risk: the seduction of common sense. JAMA 2006; 296: 1653-1656
    CrossrefPubMedGoogle Scholar
  • 5 Solomon SD, McMurray JJ, Pfeffer MA, Wittes J, Fowler R, Finn P, Anderson WF, Zauber A, Hawk E, Bertagnolli M. Cardiovascular risk associated with celecoxib in a clinical trial for colorectal adenoma prevention. N Engl J Med 2005; 352: 1071-1080
    CrossrefPubMedGoogle Scholar
  • 6 Wallace JL, McKnight W, Reuter BK, Vergnolle N. NSAID-induced gastric damage in rats: requirement for inhibition of both cyclooxygenase 1 and 2. Gastroenterology 2000; 119: 706-714
    CrossrefPubMedGoogle Scholar
  • 7 Graham DY, Agrawal NM, Roth SH. Prevention of NSAID-induced gastric ulcer with misoprostol: multicentre, double-blind, placebo-controlled trial. Lancet 1988; 2: 1277-1280
    PubMedGoogle Scholar
  • 8 Chaudhary LB, Rao RR. Taxonomic study of herbaceous species of Phyllanthus L. (Euphorbiaceae) in India. Phytotaxonomy 2002; 2: 143-162
    PubMedGoogle Scholar
  • 9 Wang M, Cheng H, Li Y, Meng L, Zhao G, Mai K. Herbs of the genus Phyllanthus in the treatment of chronic hepatitis B: observations with three preparations from different geographic sites. J Lab Clin Med 1995; 126: 350-352
    PubMedGoogle Scholar
  • 10 Yeh SF, Hong CY, Huang YL, Liu TY, Choo KB, Chou CK. Effect of an extract from Phyllanthus amarus on hepatitis B surface antigen gene expression in human hepatoma cells. Antiviral Res 1993; 20: 185-192
    PubMedGoogle Scholar
  • 11 Eldeen IMS, Seow EM, Abdullah R, Sulaiman SF. In vitro antibacterial, antioxidant, total phenolic contents and anti-HIV-1 reverse transcriptase activities of extracts of seven Phyllanthus sp. S Afr J Bot 2011; 77: 75-79
    CrossrefPubMedGoogle Scholar
  • 12 Tang YQ, Jaganath IB, Sekaran SD. Phyllanthus spp. induces selective growth inhibition of PC-3 and MeWo human cancer cells through modulation of cell cycle and induction of apoptosis. PLoS One 2010; 5: e12644
    CrossrefPubMedGoogle Scholar
  • 13 Calixto JB, Santos AR, Cechinel Filho V, Yunes RA. A review of the plants of the genus Phyllanthus: their chemistry, pharmacology, and therapeutic potential. Med Res Rev 1998; 18: 225-258
    CrossrefPubMedGoogle Scholar
  • 14 Mali SM, Sinnathambi A, Kapase CU, Bodhankar SL, Mahadik KR. Anti-arthritic activity of standardised extract of Phyllanthus amarus in Freundʼs complete adjuvant induced arthritis. Biomed Aging Path 2011; 1: 185-190
    PubMedGoogle Scholar
  • 15 Kang MH, Kim TS, Park CG, Park HW, Ju YW, Cho YM, Koh SB, Seong HJ. Preventive effects of Phyllanthus urinaria extract on paw edema in adjuvant-induced arthritis rats. FASEB J 2008; 22: 889.5
    PubMedGoogle Scholar
  • 16 Kiemer AK, Hartung T, Huber C, Vollmar AM. Phyllanthus amarus has anti-inflammatory potential by inhibition of iNOS, COX-2, and cytokines via the NF-kappaB pathway. J Hepatol 2003; 38: 289-297
    CrossrefPubMedGoogle Scholar
  • 17 Sumantran VN, Kulkarni A, Chandwaskar R, Harsulkar A, Patwardhan B, Chopra A, Wagh UV. Chondroprotective potential of fruit extracts of Phyllanthus emblica in osteoarthritis. Evid Based Complement Alternat Med 2008; 5: 329-335
    CrossrefPubMedGoogle Scholar
  • 18 Chirdchupunseree H, Pramyothin P. Protective activity of phyllanthin in ethanol-treated primary culture of rat hepatocytes. J Ethnopharmacol 2010; 128: 172-176
    CrossrefPubMedGoogle Scholar
  • 19 Pradit W, Chomdej S, Nganvongpanit K, Ongchai S. Chondroprotective potential of Phyllanthus amarus Schum. & Thonn. in experimentally induced cartilage degradation in the explants culture model. In Vitro Cell Dev Biol Anim 2015; 51: 336-344
    CrossrefPubMedGoogle Scholar
  • 20 Kassuya CA, Leite DF, de Melo LV, Rehder VL, Calixto JB. Anti-inflammatory properties of extracts, fractions and lignans isolated from Phyllanthus amarus . Planta Med 2005; 71: 721-726
    Article in Thieme ConnectPubMedGoogle Scholar
  • 21 Hardingham TE. The role of link-protein in the structure of cartilage proteoglycan aggregates. Biochem J 1979; 177: 237-247
    CrossrefPubMedGoogle Scholar
  • 22 Felisaz N, Boumediene K, Ghayor C, Herrouin JF, Bogdanowicz P, Galerra P, Pujol JP. Stimulating effect of diacerein on TGF-beta1 and beta2 expression in articular chondrocytes cultured with and without interleukin-1. Osteoarthritis Cartilage 1999; 7: 255-264
    CrossrefPubMedGoogle Scholar
  • 23 Ong-Chai S, Chaiwongsa R, Viriyakhasem N, Pompimon W, Tangyuenyong S. Effect of active compounds from Andrographis paniculata (Nees) on protection of equine articular cartilage degradation in vitro . KKU Vet J 2008; 18: 81-96
    PubMedGoogle Scholar
  • 24 Chaiwongsa R, Ongchai S, Tangyuenyong S, Kongtawelert P, Panthong A, Reutrakul V. Chondroprotective potential of bioactive compounds of Zingiber cassumunar Roxb. against cytokine-induced cartilage degradation in explant culture. J Med Plants Res 2010; 6: 5204-5213
    PubMedGoogle Scholar
  • 25 Phitak T, Pothacharoen P, Settakorn J, Poompimol W, Caterson B, Kongtawelert P. Chondroprotective and anti-inflammatory effects of sesamin. Phytochemistry 2012; 80: 77-88
    CrossrefPubMedGoogle Scholar
  • 26 Goranov NV. Serum markers of lipid peroxidation, antioxidant enzymatic defense, and collagen degradation in an experimental (Pond-Nuki) canine model of osteoarthritis. Vet Clin Pathol 2007; 36: 192-195
    CrossrefPubMedGoogle Scholar
  • 27 Clark IM, Powell LK, Ramsey S, Hazleman BL, Cawston TE. The measurement of collagenase, tissue inhibitor of metalloproteinases (TIMP), and collagenase-TIMP complex in synovial fluids from patients with osteoarthritis and rheumatoid arthritis. Arthritis Rheum 1993; 36: 372-379
    CrossrefPubMedGoogle Scholar
  • 28 Toyoshima T, Matsushita O, Minami J, Nishi N, Okabe A, Itano T. Collagen-binding domain of a Clostridium histolyticum collagenase exhibits a broad substrate spectrum both in vitro and in vivo . Connect Tissue Res 2001; 42: 281-290
    CrossrefPubMedGoogle Scholar
  • 29 Barla F, Higashijima H, Funai S, Sugimoto K, Harada N, Yamaji R, Fujita T, Nakano Y, Inui H. Inhibitive effects of alkyl gallates on hyaluronidase and collagenase. Biosci Biotechnol Biochem 2009; 73: 2335-2337
    CrossrefPubMedGoogle Scholar
  • 30 Sin BY, Kim HP. Inhibition of collagenase by naturally-occurring flavonoids. Arch Pharm Res 2005; 28: 1152-1155
    CrossrefPubMedGoogle Scholar
  • 31 Natarajan V, Madhan B, Tiku ML. Intra-articular injections of polyphenols protect articular cartilage from inflammation-induced degradation: suggesting a potential role in cartilage therapeutics. PLoS One 2015; 10: e0127165
    CrossrefPubMedGoogle Scholar
  • 32 Murugaiyah V, Chan KL. Determination of four lignans in Phyllanthus niruri L. by a simple high-performance liquid chromatography method with fluorescence detection. J Chromatogr A 2007; 1154: 198-204
    CrossrefPubMedGoogle Scholar
  • 33 Körner C. Alpine Plant Life. Berlin: Springer; 1999
    CrossrefGoogle Scholar
  • 34 Tripathi AK, Verma RK, Gupta AK, Gupta MM, Khanuja SP. Quantitative determination of phyllanthin and hypophyllanthin in Phyllanthus species by high-performance thin layer chromatography. Phytochem Anal 2006; 17: 394-397
    CrossrefPubMedGoogle Scholar
  • 35 Srivastava V, Singh M, Malasoni R, Shanker K, Verma RK, Gupta MM, Gupta AK, Khanuja SP. Separation and quantification of lignans in Phyllanthus species by a simple chiral densitometric method. J Sep Sci 2008; 31: 47-55
    CrossrefPubMedGoogle Scholar
  • 36 Escarpa A, Gonzalez MC. High-performance liquid chromatography with diode-array detection for the determination of phenolic compounds in peel and pulp from different apple varieties. J Chromatogr A 1998; 823: 331-337
    CrossrefPubMedGoogle Scholar
  • 37 Narasimhudu CL, Raju RV. Preliminary phytochemical studies on leaves of Phyllanthus species (Euphorbiaceae), used by the local tribals of Andhra Pradesh. Int J Pharmaceut Stud Res 2011; 2: 6-9
    PubMedGoogle Scholar
  • 38 Sarin B, Verma N. An overview of important ethnomedicinal herbs of Phyllanthus species: present status and future prospects. ScientificWorldJournal 2014; 2014: 839172
    PubMedGoogle Scholar
  • 39 Kim YJ, Uyama H, Kobayashi S. Inhibition effects of (+)-catechin-aldehyde polycondensates on proteinases causing proteolytic degradation of extracellular matrix. Biochem Biophys Res Commun 2004; 320: 256-261
    CrossrefPubMedGoogle Scholar
  • 40 Madhan B, Krishnamoorthy G, Rao JR, Nair BU. Role of green tea polyphenols in the inhibition of collagenolytic activity by collagenase. Int J Biol Macromol 2007; 41: 16-22
    CrossrefPubMedGoogle Scholar
  • 41 Demeule M, Brossard M, Page M, Gingras D, Beliveau R. Matrix metalloproteinase inhibition by green tea catechins. Biochim Biophys Acta 2000; 1478: 51-60
    CrossrefPubMedGoogle Scholar
  • 42 Ahmed S, Wang N, Hafeez BB, Cheruvu VK, Haqqi TM. Punica granatum L. extract inhibits IL-1beta-induced expression of matrix metalloproteinases by inhibiting the activation of MAP kinases and NF-kappaB in human chondrocytes in vitro . J Nutr 2005; 135: 2096-2102
    CrossrefPubMedGoogle Scholar
  • 43 Van Welzen PC, Chayamarit K. Flora of Thailand Euphorbiaceae. Naturalis Biodiversity Center, Leiden; Forest Herbarium, National Park, Wildlife and Plant Conservation Department, Bangkok. Available at. http://www.nationaalherbarium.nl/thaieuph Accessed August 20, 2014
    PubMedGoogle Scholar
  • 44 Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to evaluate antioxidant activity. LWT Food Sci Technol 1995; 28: 25-30
    CrossrefPubMedGoogle Scholar
  • 45 Chandler SF, Dodds JH. The effect of phosphate, nitrogen and sucrose on the production of phenolics and solasodine in callus cultures of solanum laciniatum. Plant Cell Rep 1983; 2: 205-208
    CrossrefPubMedGoogle Scholar
  • 46 Chang CC, Yang MH, Wen HM, Chern JC. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J Food Drug Anal 2002; 10: 178-182
    PubMedGoogle Scholar
  • 47 Osathanunkul M, Buddhachat K, Chomdej S. A modified colorimetric method of gelatinolytic assay using bacterial collagenase type II as a model. Anal Biochem 2013; 433: 168-170
    CrossrefPubMedGoogle Scholar
  • 48 Douni E, Sfikakis PP, Haralambous S, Fernandes P, Kollias G. Attenuation of inflammatory polyarthritis in TNF transgenic mice by diacerein: comparative analysis with dexamethasone, methotrexate and anti-TNF protocols. Arthritis Res Ther 2004; 6: R65-R72
    CrossrefPubMedGoogle Scholar
  • 49 Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta 1986; 883: 173-177
    CrossrefPubMedGoogle Scholar
  • 50 Cesaretti M, Luppi E, Maccari F, Volpi N. A 96-well assay for uronic acid carbazole reaction. Carbohydr Polym 2003; 54: 59-61
    CrossrefPubMedGoogle Scholar
  • 51 Huh JE, Baek YH, Kim YJ, Lee JD, Choi DY, Park DS. Protective effects of butanol fraction from Betula platyphyla var. japonica on cartilage alterations in a rabbit collagenase-induced osteoarthritis. J Ethnopharmacol 2009; 123: 515-521
    CrossrefPubMedGoogle Scholar