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Phyllanthus spp. as a Potential Alternative Treatment for Arthritic Conditions
CHAPTER 30
Phyllanthus spp. as a Potential Alternative Treatment for Arthritic Conditions Siriwan Ongchai Thailand Excellence Center for Tissue Engineering and Stem Cells, Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
ABBREVIATIONS IFN IL LPS MMP OA RA TNF
interferon interleukin lipopolysaccharides matrix metalloproteinase osteoarthritis rheumatoid arthritis tumor necrosis factor
1. INTRODUCTION Genus Phyllanthus, a member of the flowering plant family Phyllanthaceae, includes more than 1000 species that are widely distributed in the tropical and subtropical zones of Asia, Africa, Australia, and South America.1 Several herbaceous Phyllanthus species have been reported to contain secondary metabolites with a wide range of pharmacological properties.2 P. amarus (Figure 30.1), P. urinaria, P. niruri, and P. emblica3–5 have been used since ancient times to treat urinary diseases, constipation, hypertension, fever, respiratory inflammation, muscle pain, diarrhea, gallbladder disease, urinary disorders,6 sexually transmitted diseases,7 diabetes,8 wounds,9 rheumatism,10 and arthritis11. Arthritis includes joint disorders characterized by inflammation, pain, joint stiffness, deterioration of joint cartilage and surrounding structures, and decreased range of motion.12,13 Osteoarthritis (OA) and rheumatoid arthritis (RA) are two of the most common musculoskeletal disorders.12 Although the primary symptoms of both OA and RA are inflammation and joint damage, they differ in pathogenesis. OA is a noninfectious degenerative joint disease with chronic low-grade inflammation.13 Suppression of cartilage anabolism and repair concurrent with acceleration of cartilage catabolism lead to chondrocyte hypertrophy and apoptosis. Disease progression involves the activation of inflammatory cytokines and chemokines, adipokines, Bioactive Food as Dietary Interventions for Arthritis and Related Inflammatory Diseases https://doi.org/10.1016/B978-0-12-813820-5.00030-1
© 2019 Elsevier Inc. All rights reserved.
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Bioactive Food as Dietary Interventions for Arthritis and Related Inflammatory Diseases
Figure 30.1 Phyllanthus amarus Schumach. & Thonn.
prostaglandins, nitric oxide, etc. The expression and activity of enzymes that degrade cartilage matrix proteins, especially collagen and aggrecan, are upregulated.14,15 These conditions lead to a gradual loss of cartilage matrix, osteophyte formation, and inflammation of the synovium.16,17 RA is a chronic autoimmune disorder characterized by joint destruction and disability, and with an inflammatory progression distinct from that of OA.18 Joints affected by RA are often warm, swollen, and painful.19 Fibroblast-like synoviocytes, which are predominant in the synovial intima of joints affected by RA, produce high levels of cytokines and growth factors that lead to infiltration of inflammatory cells.14 These events lead to other inflammation-related conditions and metabolic changes.15 Fibroblast-like synoviocytes are resistant to apoptosis,16 and along with chondrocytes, the resident cells of cartilage tissue, are activated within the affected joints. The resulting increased expression of proinflammatory cytokines, nitric oxide, prostaglandin E2, and matrix-degrading enzymes contributes to joint inflammation and acceleration of cartilage degeneration.17 Nitric oxide is known as a catabolic factor that contributes to the pathogenesis of arthritis.20 High concentrations of both nitric oxide and proinflammatory cytokines coexist in the synovial fluid and plasma of RA and OA21 patients. Some studies reported that nitric oxide increased inflammation and pain perception,22 while others have described the protective effects of nitric oxide on various cell types, including chondrocytes.23 Drugs that suppress the pathogenesis of OA and RA target reduction of the inflammation that underlies pain, joint swelling, and other symptoms. Frequent side effects of antiarthritis drugs involve injury of the gastrointestinal tract, especially in elderly people.24 Herbal medicines are an increasingly popular treatment alternative, and the evidence in support of their antiarthritic effectiveness is increasing. As shown in Figure 30.2, the herbaceous Phyllanthus species are reported to have antiinflammatory, antinociceptive, antioxidative, and chondroprotective properties.
Phyllanthus spp. as a Potential Alternative Treatment
Figure 30.2 Biological activities that contribute to the antiarthritic potential of Phyllanthus.
2. METHODS OF EVALUATING ANTIARTHRITIC PROPERTIES IN VITRO AND IN VIVO Several in vitro and in vivo models have been used to evaluate the antiarthritic potential of Phyllanthus spp. The lipopolysaccharide (LPS)-stimulated production of proinflammatory factors in several monocytic cell cultures has been used in in vitro.25 These systems are also acceptable for investigation of intracellular molecular mechanisms. Animal models include inflammatory agent-induced paw edema and cotton pelletinduced granuloma formation to evaluate acute and subacute inflammation. The analgesic properties26,27 of test compounds have been evaluated in animal models such as hot plate and tail flick responses, acetic acid-induced writhing, and formalin-induced pain.28 Freund’s complete adjuvant-induced arthritis can be used to model and evaluate chronic systemic inflammation with progressive joint destruction similar RA.29 Cartilage explant culture models of joint degradation stimulated by proinflammatory cytokines have been used to evaluate the chondroprotection of Phyllanthus in vitro.30,31 Chondrocyte culture systems with exposure to proinflammatory cytokines are frequently used to investigate protective mechanisms at the molecular level.32 The evidence obtained from these experimental models provides early preclinical support of Phyllanthus in arthritis therapy.
3. ANTIARTHRITIC POTENTIAL OF PHYLLANTHUS Inflammation plays a key role in the pathogenesis of arthritis, and herbal medicines with antiinflammatory properties such as Phyllanthus have long been used to treat it. Phyllanthus is the main component of popular Ayurvedic formulations used to treat many diseases, including arthritis.11 Reports of the biological activities that support the
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Table 30.1 Phyllanthus extracts, phytochemicals, and biological activities with potential usefulness as antiarthritis treatments Biological activities with potential usefulness for antiarthritis Extract/class Phytochemical
Antiinflammation
Antinociception
Antioxidation
Hexane, ethanolic extracts31,33,34 Standardized aqueous extract35 Lignan
Flavonoid Hydroalcoholic extracts38,39 Ethanolic extract34 Standardized aqueous extract35 Sterol Triterpene Ethanolic extract34 Lignan Flavonoid Tannin Triterpene
Chondroprotection
Coumarin Ethanolic extracts45 Lignan
Phyllanthin36 Niranthin, phyltetralin, and nirtetralin33 Phyltetralin37 Rhamnocitrin, quercetin, and rutin37
β-Sitosterol40 β-Amyrin41 Phyllanthin42 Justicidin B, diphyllin43 Rutin and quercetin 3-O-glucoside44 Rhamnocitrin37 Amariin, repandusinic acid A, corilagin, and phyllanthusiin A, B, C, D44 Trimethyl 1–3,4 dehydrochebulate and methyl gallate37 Methylbrevifolin carboxylate37 Phyllanthin and hypophyllanthin46
antiarthritic activity of Phyllanthus and their phytochemical components are summarized in Figure 30.2. The phytochemicals isolated from members of the genus Phyllanthus include lignans, flavonoids, tannins, alkaloids, sterols, and triterpenes.2 Those with antiarthritic activity are shown in Table 30.1.
3.1 P. amarus Schumach. & Thonn. Kiemer et al. were the first to report the in vitro and in vivo antiinflammatory activities of the ethanolic and hexane extracts of P. amarus.25 These extracts suppressed the LPS induction of the proinflammatory cytokines and mediators, nitric oxide, prostaglandin
Phyllanthus spp. as a Potential Alternative Treatment
E2, and tumor necrosis factor (TNF)-α by inhibiting nuclear factor-κB signaling. The antiinflammatory activities of lignan-rich fractions and lignans isolated from P. amarus47 have been assessed by oral administration in animal models. A hexane extract, or phyltetralin, nirtetralin, and niranthin, but not hypophyllanthin or phyllanthin, ameliorated acute paw edema induced by platelet-activating factor or endothelin-1. Other P. amarus phytochemicals with potent antiinflammatory activity include ethyl 8-hydroxy-8-methyl-tridecanoate, 1,7,8-trihydroxy-2-naphtaldehyde, corilagin, and geraniin.48 Ofuegbe et al. confirmed the antiinflammatory activity of an aqueous leaf extract of P. amarus in a rat model of carrageenan- and histamine-induced paw edema.49 Its analgesic activity was evaluated by the acetic acid writhing and formalin tests. The antiarthritic activity of P. amarus was demonstrated by its effect on inflammatory parameters in a Freund’s complete adjuvant-induced rat arthritis model.46 The standardized aqueous extract significantly reduced arthritis scores, paw volume, and joint diameter, accompanied by an increase in mechanical hyperalgesia and the nociceptive threshold. The chondroprotection of P. amarus was evaluated in vitro in a cartilage explant culture model by evaluating the effect of ethanolic extract on the release of matrix metalloproteinase (MMP)-2, a cartilage-degrading enzyme, induced by the proinflammatory cytokine interleukin (IL)-1β.30 This assay revealed weak, dose-dependent chondroprotective activity of the reference standards, phyllanthin and hypophyllanthin, the major phytochemicals of P. amarus. The results suggested that those lignans might not be the only P. amarus components responsible for its chondroprotective properties. In the same cartilage explant culture model, Buddhachat et al. reported that P. amarus, P. urinaria, P. debilis, and P. airy-shawii had similar chondroprotective properties.31 Investigations utilizing chondrocyte cell culture models demonstrated Phyllanthus protective activity at the molecular level. As shown in Figure 30.3, IL-1β downregulation of the expression of the SOX9 transcription factor gene (encoding protein transcription factor SOX-9) and the COL2A1 gene (encoding type II collagen) was restored to normal by the polar rich fraction (PRF) of P. amarus. The transcription factor SOX-9 plays an important role in upregulation of the expression of key cartilaginous matrix genes, including those encoding collagen type II and aggrecan.45 PRF significantly suppressed the mRNA expression of TNF-α and MMP13 induced by IL-1β. The translation products are catabolic factors associated with degenerative arthritis. These results suggest that the beneficial properties of P. amarus are both chondroprotection and activation of the synthesis of cartilage biomolecules that are degraded under the inflammatory condition.
3.2 P. emblica L. P. emblica fruit is used as a herbal medicine for pain relief and to attenuate inflammatory conditions. Jaijoy et al. reported the potent antiinflammatory activity of a water extract of
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Figure 30.3 The polar rich fraction (PRF) of the ethanolic extract of P. amarus (25, 50, 100 μg/mL) attenuates the effects of IL-1β (10 ng/mL) either upregulating SOX9 and COL2A1 mRNA expression, or suppressing MMP13 and TNF-α mRNA expression in a SW1353 chondrocyte cell culture model. Ctrl, control; DIAC, diacerein 50 μM, *P < 0.05, **P < 0.01 (S. Ongchai, unpublished data).
Phyllanthus spp. as a Potential Alternative Treatment
P. emblica fruit by its significant reduction of carrageenan-induced paw edema in rats.50 The formalin test confirmed the analgesic activity of the water extract following oral administration in mice, in both early and late phases edema. The activity of the water extract was similar to that of nonsteroidal antiinflammatory drugs. The chondroprotective activity of an aqueous extract of unprocessed P. emblica fruit was reported33 in vitro in short- and long-term explant cultures of articular knee cartilage from osteoarthritis patients. The extract inhibited hyaluronidase and collagenase type 2 activity, which are associated with cartilage degradation, and inhibited the release of glycosaminoglycan from cartilage tissue. The antiinflammatory effects of P. emblica were confirmed in mouse lung tissue with benzo(a)pyrene-induced increase in TNF-α, IL-6, and IL-1β proinflammatory cytokines.34 The antiinflammatory activity of polyherbal formulations containing P. emblica was investigated in a carrageenan-induced rat paw edema and cotton-pellet granuloma formation.35 The therapeutic efficacy to reduce pain and improve the quality of life of formulations containing P. emblica extract was reported in moderately arthritic dogs.36
3.3 P. acidus (L.) Skeels Chakraborty et al. investigated the antiinflammatory and analgesic activities of methanol, ethyl acetate, and petroleum ether extracts of P. acidus leaves.37 The methanol extract had the strongest antiinflammatory activity in rat models of carrageenan-induced paw edema and cotton pellet-induced granuloma formation. The methanol extract also had the best analgesic activity in the early and late phases of the formalin test. The results indicated that the antiinflammatory, analgesic, and antioxidant activities were correlated with the flavonoid and phenolic content of the extract. Similar results were confirmed in models using Swiss albino mice.38 The antiinflammatory activity of a P. acidus methanol extract was verified in vitro in LPS-treated RAW264.7 cell cultures.39 The extract suppressed the LPS-induced expression of inducible nitric oxide synthase and cyclooxygenase-2 via suppression of nuclear factor-κB signaling pathway activation. Two phytochemicals in the extract, kaempferol and quercetin, partially accounted for the antiinflammatory activity.
3.4 P. niruri L. Porto et al. investigated the antiinflammatory and antinociceptive potential of P. niruri in adult albino rats and mice.40 In the mouse model, the intraperitoneal injection of a spraydried standardized extract of P. niruri reduced the vascular response in carrageenaninduced paw edema. Oral administration inhibited leukocyte migration induced by 3% thioglycolate. Intraperitoneal injection of the extract in a rat model had a marked peripheral and central analgesic effect in the Randall and Selitto assay and the hot plate
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and tail flick tests. The antioxidant activity of P. niruri was evaluated in healthy nonsmoking male volunteers.41 After drinking P. niruri tea, plasma ascorbic and gallic acid were increased at 1 and 4 h. Erythrocytic catalase and superoxide dismutase activities were not changed. These data provide evidence of the antioxidation activity of oral P. niruri in humans, which contributes to its pharmacological properties.
3.5 P. muellerianus (Kuntze) Exell. P. muellerianus, a medicinal plant that grows in West Africa, has long been used to treat healing wounds, fevers, pain, and inflammation. The antiinflammatory properties of P. muellerianus have been reported in experimentally induced inflammation in rats.42 An aqueous leaf extract and its major isolate, geraniin, attenuated adjuvant-induced chronic inflammation, with reduction of hind-limb swelling and bone damage. The results are in line with a previous report of P. muellerianus active constituents in promoting collagen synthesis in dermal fibroblasts.42 Both studies are consistent with activation of the synthesis of cartilage extracellular matrix molecules that is reduced in the low-grade and chronic inflammation of arthritis.
3.6 P. polyphyllus Willd., P. urinaria L., P. debilis Klein ex Willd., and P. kozhikodianus Sivar. & Manilal Rao et al. demonstrated the antiinflammatory properties of four phytochemicals isolated from P. polyphyllus, including benzenoid, 4-O-methylgallic acid, and three arylnaphthalide lignans, phyllamyricin C, justicidin B, and diphyllin.43 Justicidin B and diphyllin strongly reduced the production of nitric oxide and proinflammatory cytokines, TNF-α, and IL-12 in LPS- or interferon (IFN)γ-induced murine peritoneal macrophages. Fang et al. described the in vitro antioxidant and antiinflammatory activities of an ethanolic extract of P. urinaria and its phytochemicals, trimethyl-3,4-dehydrochebulate, methylgallate, and methyl brevifolincarboxylate.44 The three compounds were strong antioxidants, and together with other phytochemicals, including phyllanthin, phyltetralin, rhamnocitrin, quercitrin, and rutin, had antiinflammatory activity, suppressing production of proinflammatory markers in LPS- or IFNγ-activated peritoneal macrophages. A petroleum ether extract of P. debilis was found to reduce acute and chronic inflammation in a carrageenan-induced rat paw edema and chronic granuloma formation. Analgesic activity in the tail flick test was poor.51 Relatively few data on the biological activity of P. kozhikodianus and P. airy-shawii are available. The chondroprotection of P. airy-shawii, P. urinaria, P. debilis, and P. amarus was evaluated in vitro in a cartilage explant culture model of proinflammatory cytokineinduced cartilage degradation.31 The chondroprotective activities of these plants were not significantly different, but P. airy-shawii had the greatest antioxidant and collagenase
Phyllanthus spp. as a Potential Alternative Treatment
inhibitory activities. The total phenol and total flavonoid contents were correlated with antioxidant activity. The total phenol content was correlated with anticollagenase activity.
4. CONCLUSION Experimental evidence provides strong support for the antiinflammatory, antinociception, antioxidant, and chondroprotective activities of Phyllanthus spp. These properties contribute to their potential as an alternative arthritic therapy.
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19. Lee YC. Effect and treatment of chronic pain in inflammatory arthritis. Curr Rheumatol Rep. 2013;15(1):300. 20. Pelletier JP, Martel-Pelletier J, Abramson SB. Osteoarthritis, an inflammatory disease: potential implication for the selection of new therapeutic targets. Arthritis Rheum. 2001;44(6):1237–1247. 21. Farrell AJ, Blake DR, Palmer RM, Moncada S. Increased concentrations of nitrite in synovial fluid and serum samples suggest increased nitric oxide synthesis in rheumatic diseases. Ann Rheum Dis. 1992;51 (11):1219–1222. 22. Abramson SB. Nitric oxide in inflammation and pain associated with osteoarthritis. Arthritis Res Ther. 2008;10(suppl 2):S2. 23. Olivera GC, Ren X, Vodnala SK, et al. Nitric oxide protects against infection-induced neuroinflammation by preserving the stability of the blood-brain barrier. PLoS Pathog. 2016;12(2):e1005442. 24. Buffum M, Buffum JC. Nonsteroidal anti-inflammatory drugs in the elderly. Pain Manag Nurs. 2000;1(2):40–50. 25. 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 (3):289–297. 26. Cai C, Chen Y, Zhong S, et al. Anti-inflammatory activity of N-butanol extract from Ipomoea stolonifera in vivo and in vitro. PLoS ONE. 2014;9(4):e95931. 27. Ashok P, Koti BC, Thippeswamy AH, Tikare VP, Dabadi P, Viswanathaswamy AH. Evaluation of antiinflammatory activity of Centratherum anthelminticum (L) Kuntze seed. Indian J Pharm Sci. 2010;72 (6):697–703. 28. Ganeshpurkar A, Rai G. Experimental evaluation of analgesic and anti-inflammatory potential of Oyster mushroom Pleurotus florida. Indian J Pharmacol. 2013;45(1):66–70. 29. Osterman T, Kippo K, Lauren L, Hannuniemi R, Sellman R. Effect of clodronate on established adjuvant arthritis. Rheumatol Int. 1994;14(4):139–147. 30. 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(4):336–344. 31. Buddhachat K, Chomdej S, Pradit W, Nganvongpanit K, Ongchai S. In Vitro chondroprotective potential of extracts obtained from various Phyllantus species. Planta Med. 2017;83(1-02):87–96. 32. Ruangsuriya J, Budprom P, Viriyakhasem N, et al. Suppression of cartilage degradation by Zingerone involving the p38 and JNK MAPK signaling pathway. Planta Med. 2017;83(3-04):268–276. 33. Sumantran VN, Kulkarni A, Chandwaskar R, et al. Chondroprotective potential of fruit extracts of Phyllanthus emblica in osteoarthritis. Evid Based Complement Alternat Med. 2008;5(3):329–335. 34. Wang CC, Yuan JR, Wang CF, et al. Anti-inflammatory effects of Phyllanthus emblica L on benzopyrene-induced precancerous lung lesion by regulating the IL-1beta/miR-101/Lin28B signaling pathway. Integr Cancer Ther. 2017;16(4):505–515. 35. Deorukhakar SR, Dethe A, Vohra RR, Ghosh R, Chaudhary J, Kadam V. Antiinflammatory activity of a polyherbal formulation. Indian J Pharm Sci. 2008;70(6):785–787. 36. Gupta R. Anti-arthritic efficacy and safety of crominex 3 +(Trivalent chromium, Phyllanthus emblica extract, and shilajit) in moderately arthritic dogs. J Vet Sci Anim Husb. 2013;1:401. 37. Chakraborty R, Biplab D, Devanna N, Sen S. Antiinflammatory, antinociceptive and antioxidant activities of Phyllanthus acidus L. extracts. Asian Pac J Trop Biomed. 2012;2(2 suppl):S953–S961. 38. Hossain MS, Akter S, Begum Y, Bulbul IJ. Analgesic and anti-inflammatory activities of ethanolic leaf extract of Phyllanthus acidus L. on Swiss Albino mice. Eur J Med Plants. 2016;13(1):1. 39. Hossen MJ, Jeon SH, Kim SC, et al. In vitro and in vivo anti-inflammatory activity of Phyllanthus acidus methanolic extract. J Ethnopharmacol. 2015;168:217–228. 40. Porto CRC, Soares LAL, Souza TP, et al. Anti-inflammatory and antinociceptive activities of Phyllanthus niruri spray-dried standardized extract. Rev Bras Farmacogn. 2013;23(1):138–144. 41. Colpo E, Vilanova CD, Pereira RP, et al. Antioxidant effects of Phyllanthus niruri tea on healthy subjects. Asian Pac J Trop Med. 2014;7(2):113–118. 42. Agyare C, Lechtenberg M, Deters A, Petereit F, Hensel A. Ellagitannins from Phyllanthus muellerianus (Kuntze) Exell.: geraniin and furosin stimulate cellular activity, differentiation and collagen synthesis of human skin keratinocytes and dermal fibroblasts. Phytomedicine. 2011;18(7):617–624.
Phyllanthus spp. as a Potential Alternative Treatment
43. Rao YK, Fang SH, Tzeng YM. Anti-inflammatory activities of constituents isolated from Phyllanthus polyphyllus. J Ethnopharmacol. 2006;103(2):181–186. 44. Fang SH, Rao YK, Tzeng YM. Anti-oxidant and inflammatory mediator’s growth inhibitory effects of compounds isolated from Phyllanthus urinaria. J Ethnopharmacol. 2008;116(2):333–340. 45. Henry SP, Liang S, Akdemir KC, de Crombrugghe B. The postnatal role of Sox9 in cartilage. J Bone Miner Res. 2012;27(12):2511–2525. 46. Mali SM, Sinnathambi A, Kapase CU, Bodhankar SL, Mahadik KR. Anti-arthritic activity of standardised extract of Phyllanthus amarus in Freund’s complete adjuvant induced arthritis. Biomed Aging Pathol. 2011;1(3):185–190. 47. 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(8):721–726. 48. Yuandani JI, Ilangkovan M, Husain K, Chan KM. Inhibitory effects of compounds from Phyllanthus amarus on nitric oxide production, lymphocyte proliferation, and cytokine release from phagocytes. Drug Des Devel Ther. 2016;10:1935–1945. 49. Adedapo AA, Ofuegbe SO. Anti-inflammatory and antinociceptive activities of the aqueous leaf extract of Phyllanthus amarus Schum (Euphorbiaceae) in some laboratory animals. J Basic Clin Physiol Pharmacol. 2015;26(1):89–94. 50. Jaijoy K, Soonthornchareonnon N, Panthong A, Sireeratawong S. Anti-inflammatory and analgesic activities of the water extract from the fruit of Phyllanthus emblica Linn. Int J Appl Res Nat Prod. 2010;3(2):8. 51. Chandrashekar KS, Joshi AB, Satyanarayana D, Pai P. Analgesic and anti-inflammatory activities of Phyllanthus debilis. Whole plant. Pharm Biol. 2005;43(7):586–588.
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Phyllanthus spp. as a Potential Alternative Treatment for Arthritic Conditions Siriwan Ongchai Thailand Excellence Center for Tissue Engineering and Stem Cells, Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
ABBREVIATIONS IFN IL LPS MMP OA RA TNF
interferon interleukin lipopolysaccharides matrix metalloproteinase osteoarthritis rheumatoid arthritis tumor necrosis factor
1. INTRODUCTION Genus Phyllanthus, a member of the flowering plant family Phyllanthaceae, includes more than 1000 species that are widely distributed in the tropical and subtropical zones of Asia, Africa, Australia, and South America.1 Several herbaceous Phyllanthus species have been reported to contain secondary metabolites with a wide range of pharmacological properties.2 P. amarus (Figure 30.1), P. urinaria, P. niruri, and P. emblica3–5 have been used since ancient times to treat urinary diseases, constipation, hypertension, fever, respiratory inflammation, muscle pain, diarrhea, gallbladder disease, urinary disorders,6 sexually transmitted diseases,7 diabetes,8 wounds,9 rheumatism,10 and arthritis11. Arthritis includes joint disorders characterized by inflammation, pain, joint stiffness, deterioration of joint cartilage and surrounding structures, and decreased range of motion.12,13 Osteoarthritis (OA) and rheumatoid arthritis (RA) are two of the most common musculoskeletal disorders.12 Although the primary symptoms of both OA and RA are inflammation and joint damage, they differ in pathogenesis. OA is a noninfectious degenerative joint disease with chronic low-grade inflammation.13 Suppression of cartilage anabolism and repair concurrent with acceleration of cartilage catabolism lead to chondrocyte hypertrophy and apoptosis. Disease progression involves the activation of inflammatory cytokines and chemokines, adipokines, Bioactive Food as Dietary Interventions for Arthritis and Related Inflammatory Diseases https://doi.org/10.1016/B978-0-12-813820-5.00030-1
© 2019 Elsevier Inc. All rights reserved.
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Figure 30.1 Phyllanthus amarus Schumach. & Thonn.
prostaglandins, nitric oxide, etc. The expression and activity of enzymes that degrade cartilage matrix proteins, especially collagen and aggrecan, are upregulated.14,15 These conditions lead to a gradual loss of cartilage matrix, osteophyte formation, and inflammation of the synovium.16,17 RA is a chronic autoimmune disorder characterized by joint destruction and disability, and with an inflammatory progression distinct from that of OA.18 Joints affected by RA are often warm, swollen, and painful.19 Fibroblast-like synoviocytes, which are predominant in the synovial intima of joints affected by RA, produce high levels of cytokines and growth factors that lead to infiltration of inflammatory cells.14 These events lead to other inflammation-related conditions and metabolic changes.15 Fibroblast-like synoviocytes are resistant to apoptosis,16 and along with chondrocytes, the resident cells of cartilage tissue, are activated within the affected joints. The resulting increased expression of proinflammatory cytokines, nitric oxide, prostaglandin E2, and matrix-degrading enzymes contributes to joint inflammation and acceleration of cartilage degeneration.17 Nitric oxide is known as a catabolic factor that contributes to the pathogenesis of arthritis.20 High concentrations of both nitric oxide and proinflammatory cytokines coexist in the synovial fluid and plasma of RA and OA21 patients. Some studies reported that nitric oxide increased inflammation and pain perception,22 while others have described the protective effects of nitric oxide on various cell types, including chondrocytes.23 Drugs that suppress the pathogenesis of OA and RA target reduction of the inflammation that underlies pain, joint swelling, and other symptoms. Frequent side effects of antiarthritis drugs involve injury of the gastrointestinal tract, especially in elderly people.24 Herbal medicines are an increasingly popular treatment alternative, and the evidence in support of their antiarthritic effectiveness is increasing. As shown in Figure 30.2, the herbaceous Phyllanthus species are reported to have antiinflammatory, antinociceptive, antioxidative, and chondroprotective properties.
Phyllanthus spp. as a Potential Alternative Treatment
Figure 30.2 Biological activities that contribute to the antiarthritic potential of Phyllanthus.
2. METHODS OF EVALUATING ANTIARTHRITIC PROPERTIES IN VITRO AND IN VIVO Several in vitro and in vivo models have been used to evaluate the antiarthritic potential of Phyllanthus spp. The lipopolysaccharide (LPS)-stimulated production of proinflammatory factors in several monocytic cell cultures has been used in in vitro.25 These systems are also acceptable for investigation of intracellular molecular mechanisms. Animal models include inflammatory agent-induced paw edema and cotton pelletinduced granuloma formation to evaluate acute and subacute inflammation. The analgesic properties26,27 of test compounds have been evaluated in animal models such as hot plate and tail flick responses, acetic acid-induced writhing, and formalin-induced pain.28 Freund’s complete adjuvant-induced arthritis can be used to model and evaluate chronic systemic inflammation with progressive joint destruction similar RA.29 Cartilage explant culture models of joint degradation stimulated by proinflammatory cytokines have been used to evaluate the chondroprotection of Phyllanthus in vitro.30,31 Chondrocyte culture systems with exposure to proinflammatory cytokines are frequently used to investigate protective mechanisms at the molecular level.32 The evidence obtained from these experimental models provides early preclinical support of Phyllanthus in arthritis therapy.
3. ANTIARTHRITIC POTENTIAL OF PHYLLANTHUS Inflammation plays a key role in the pathogenesis of arthritis, and herbal medicines with antiinflammatory properties such as Phyllanthus have long been used to treat it. Phyllanthus is the main component of popular Ayurvedic formulations used to treat many diseases, including arthritis.11 Reports of the biological activities that support the
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Table 30.1 Phyllanthus extracts, phytochemicals, and biological activities with potential usefulness as antiarthritis treatments Biological activities with potential usefulness for antiarthritis Extract/class Phytochemical
Antiinflammation
Antinociception
Antioxidation
Hexane, ethanolic extracts31,33,34 Standardized aqueous extract35 Lignan
Flavonoid Hydroalcoholic extracts38,39 Ethanolic extract34 Standardized aqueous extract35 Sterol Triterpene Ethanolic extract34 Lignan Flavonoid Tannin Triterpene
Chondroprotection
Coumarin Ethanolic extracts45 Lignan
Phyllanthin36 Niranthin, phyltetralin, and nirtetralin33 Phyltetralin37 Rhamnocitrin, quercetin, and rutin37
β-Sitosterol40 β-Amyrin41 Phyllanthin42 Justicidin B, diphyllin43 Rutin and quercetin 3-O-glucoside44 Rhamnocitrin37 Amariin, repandusinic acid A, corilagin, and phyllanthusiin A, B, C, D44 Trimethyl 1–3,4 dehydrochebulate and methyl gallate37 Methylbrevifolin carboxylate37 Phyllanthin and hypophyllanthin46
antiarthritic activity of Phyllanthus and their phytochemical components are summarized in Figure 30.2. The phytochemicals isolated from members of the genus Phyllanthus include lignans, flavonoids, tannins, alkaloids, sterols, and triterpenes.2 Those with antiarthritic activity are shown in Table 30.1.
3.1 P. amarus Schumach. & Thonn. Kiemer et al. were the first to report the in vitro and in vivo antiinflammatory activities of the ethanolic and hexane extracts of P. amarus.25 These extracts suppressed the LPS induction of the proinflammatory cytokines and mediators, nitric oxide, prostaglandin
Phyllanthus spp. as a Potential Alternative Treatment
E2, and tumor necrosis factor (TNF)-α by inhibiting nuclear factor-κB signaling. The antiinflammatory activities of lignan-rich fractions and lignans isolated from P. amarus47 have been assessed by oral administration in animal models. A hexane extract, or phyltetralin, nirtetralin, and niranthin, but not hypophyllanthin or phyllanthin, ameliorated acute paw edema induced by platelet-activating factor or endothelin-1. Other P. amarus phytochemicals with potent antiinflammatory activity include ethyl 8-hydroxy-8-methyl-tridecanoate, 1,7,8-trihydroxy-2-naphtaldehyde, corilagin, and geraniin.48 Ofuegbe et al. confirmed the antiinflammatory activity of an aqueous leaf extract of P. amarus in a rat model of carrageenan- and histamine-induced paw edema.49 Its analgesic activity was evaluated by the acetic acid writhing and formalin tests. The antiarthritic activity of P. amarus was demonstrated by its effect on inflammatory parameters in a Freund’s complete adjuvant-induced rat arthritis model.46 The standardized aqueous extract significantly reduced arthritis scores, paw volume, and joint diameter, accompanied by an increase in mechanical hyperalgesia and the nociceptive threshold. The chondroprotection of P. amarus was evaluated in vitro in a cartilage explant culture model by evaluating the effect of ethanolic extract on the release of matrix metalloproteinase (MMP)-2, a cartilage-degrading enzyme, induced by the proinflammatory cytokine interleukin (IL)-1β.30 This assay revealed weak, dose-dependent chondroprotective activity of the reference standards, phyllanthin and hypophyllanthin, the major phytochemicals of P. amarus. The results suggested that those lignans might not be the only P. amarus components responsible for its chondroprotective properties. In the same cartilage explant culture model, Buddhachat et al. reported that P. amarus, P. urinaria, P. debilis, and P. airy-shawii had similar chondroprotective properties.31 Investigations utilizing chondrocyte cell culture models demonstrated Phyllanthus protective activity at the molecular level. As shown in Figure 30.3, IL-1β downregulation of the expression of the SOX9 transcription factor gene (encoding protein transcription factor SOX-9) and the COL2A1 gene (encoding type II collagen) was restored to normal by the polar rich fraction (PRF) of P. amarus. The transcription factor SOX-9 plays an important role in upregulation of the expression of key cartilaginous matrix genes, including those encoding collagen type II and aggrecan.45 PRF significantly suppressed the mRNA expression of TNF-α and MMP13 induced by IL-1β. The translation products are catabolic factors associated with degenerative arthritis. These results suggest that the beneficial properties of P. amarus are both chondroprotection and activation of the synthesis of cartilage biomolecules that are degraded under the inflammatory condition.
3.2 P. emblica L. P. emblica fruit is used as a herbal medicine for pain relief and to attenuate inflammatory conditions. Jaijoy et al. reported the potent antiinflammatory activity of a water extract of
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Figure 30.3 The polar rich fraction (PRF) of the ethanolic extract of P. amarus (25, 50, 100 μg/mL) attenuates the effects of IL-1β (10 ng/mL) either upregulating SOX9 and COL2A1 mRNA expression, or suppressing MMP13 and TNF-α mRNA expression in a SW1353 chondrocyte cell culture model. Ctrl, control; DIAC, diacerein 50 μM, *P < 0.05, **P < 0.01 (S. Ongchai, unpublished data).
Phyllanthus spp. as a Potential Alternative Treatment
P. emblica fruit by its significant reduction of carrageenan-induced paw edema in rats.50 The formalin test confirmed the analgesic activity of the water extract following oral administration in mice, in both early and late phases edema. The activity of the water extract was similar to that of nonsteroidal antiinflammatory drugs. The chondroprotective activity of an aqueous extract of unprocessed P. emblica fruit was reported33 in vitro in short- and long-term explant cultures of articular knee cartilage from osteoarthritis patients. The extract inhibited hyaluronidase and collagenase type 2 activity, which are associated with cartilage degradation, and inhibited the release of glycosaminoglycan from cartilage tissue. The antiinflammatory effects of P. emblica were confirmed in mouse lung tissue with benzo(a)pyrene-induced increase in TNF-α, IL-6, and IL-1β proinflammatory cytokines.34 The antiinflammatory activity of polyherbal formulations containing P. emblica was investigated in a carrageenan-induced rat paw edema and cotton-pellet granuloma formation.35 The therapeutic efficacy to reduce pain and improve the quality of life of formulations containing P. emblica extract was reported in moderately arthritic dogs.36
3.3 P. acidus (L.) Skeels Chakraborty et al. investigated the antiinflammatory and analgesic activities of methanol, ethyl acetate, and petroleum ether extracts of P. acidus leaves.37 The methanol extract had the strongest antiinflammatory activity in rat models of carrageenan-induced paw edema and cotton pellet-induced granuloma formation. The methanol extract also had the best analgesic activity in the early and late phases of the formalin test. The results indicated that the antiinflammatory, analgesic, and antioxidant activities were correlated with the flavonoid and phenolic content of the extract. Similar results were confirmed in models using Swiss albino mice.38 The antiinflammatory activity of a P. acidus methanol extract was verified in vitro in LPS-treated RAW264.7 cell cultures.39 The extract suppressed the LPS-induced expression of inducible nitric oxide synthase and cyclooxygenase-2 via suppression of nuclear factor-κB signaling pathway activation. Two phytochemicals in the extract, kaempferol and quercetin, partially accounted for the antiinflammatory activity.
3.4 P. niruri L. Porto et al. investigated the antiinflammatory and antinociceptive potential of P. niruri in adult albino rats and mice.40 In the mouse model, the intraperitoneal injection of a spraydried standardized extract of P. niruri reduced the vascular response in carrageenaninduced paw edema. Oral administration inhibited leukocyte migration induced by 3% thioglycolate. Intraperitoneal injection of the extract in a rat model had a marked peripheral and central analgesic effect in the Randall and Selitto assay and the hot plate
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and tail flick tests. The antioxidant activity of P. niruri was evaluated in healthy nonsmoking male volunteers.41 After drinking P. niruri tea, plasma ascorbic and gallic acid were increased at 1 and 4 h. Erythrocytic catalase and superoxide dismutase activities were not changed. These data provide evidence of the antioxidation activity of oral P. niruri in humans, which contributes to its pharmacological properties.
3.5 P. muellerianus (Kuntze) Exell. P. muellerianus, a medicinal plant that grows in West Africa, has long been used to treat healing wounds, fevers, pain, and inflammation. The antiinflammatory properties of P. muellerianus have been reported in experimentally induced inflammation in rats.42 An aqueous leaf extract and its major isolate, geraniin, attenuated adjuvant-induced chronic inflammation, with reduction of hind-limb swelling and bone damage. The results are in line with a previous report of P. muellerianus active constituents in promoting collagen synthesis in dermal fibroblasts.42 Both studies are consistent with activation of the synthesis of cartilage extracellular matrix molecules that is reduced in the low-grade and chronic inflammation of arthritis.
3.6 P. polyphyllus Willd., P. urinaria L., P. debilis Klein ex Willd., and P. kozhikodianus Sivar. & Manilal Rao et al. demonstrated the antiinflammatory properties of four phytochemicals isolated from P. polyphyllus, including benzenoid, 4-O-methylgallic acid, and three arylnaphthalide lignans, phyllamyricin C, justicidin B, and diphyllin.43 Justicidin B and diphyllin strongly reduced the production of nitric oxide and proinflammatory cytokines, TNF-α, and IL-12 in LPS- or interferon (IFN)γ-induced murine peritoneal macrophages. Fang et al. described the in vitro antioxidant and antiinflammatory activities of an ethanolic extract of P. urinaria and its phytochemicals, trimethyl-3,4-dehydrochebulate, methylgallate, and methyl brevifolincarboxylate.44 The three compounds were strong antioxidants, and together with other phytochemicals, including phyllanthin, phyltetralin, rhamnocitrin, quercitrin, and rutin, had antiinflammatory activity, suppressing production of proinflammatory markers in LPS- or IFNγ-activated peritoneal macrophages. A petroleum ether extract of P. debilis was found to reduce acute and chronic inflammation in a carrageenan-induced rat paw edema and chronic granuloma formation. Analgesic activity in the tail flick test was poor.51 Relatively few data on the biological activity of P. kozhikodianus and P. airy-shawii are available. The chondroprotection of P. airy-shawii, P. urinaria, P. debilis, and P. amarus was evaluated in vitro in a cartilage explant culture model of proinflammatory cytokineinduced cartilage degradation.31 The chondroprotective activities of these plants were not significantly different, but P. airy-shawii had the greatest antioxidant and collagenase
Phyllanthus spp. as a Potential Alternative Treatment
inhibitory activities. The total phenol and total flavonoid contents were correlated with antioxidant activity. The total phenol content was correlated with anticollagenase activity.
4. CONCLUSION Experimental evidence provides strong support for the antiinflammatory, antinociception, antioxidant, and chondroprotective activities of Phyllanthus spp. These properties contribute to their potential as an alternative arthritic therapy.
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