648 Journal of Marine Science and Technology, Vol. 24, No. 3, pp (2016) DOI: /JMST

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1 68 Journl of Mrine Science nd Technology, Vol., No. 3, pp (16) DOI:.6119/JMST FACILITATING THE RELEASE OF BIONUTRIENTS FROM MORINDA CITRIFOLIA (NONI) BY CELLULASE HYDROLYSIS AND LACTIC ACID BACTERIA FERMENTATION AND THEIR EFFECTS ON -AMYLASE AND -GLUCOSIDASE ACTIVITIES Hui-Hung Lee 1, Li-Jung Yin, Hsueh-Ming Ti 3, nd Shnn-Tzong Jing 1, Key words: Morind citrifoli, hydrolysis,,. ABSTRACT For fcilitting the relese of ionutrients from noni, 6 h of hydrolysis with U/mL cellulse t C nd n dditionl 8 h of fermenttion with Pediococcus pentosceus BCRC 153 t 37 C were performed. Increses in the extrction yields ( mg/g) nd the totl phenolic content (.-7.5 mg/g) of vrious extrcts were otined, suggesting tht ionutrients were relesed from noni during hydrolysis or fermenttion. In hydrolyzed nd/or dditionlly fermented smples, significnt decreses in the hlf mximl inhiitory concentrtion (IC ) ginst α-mylse ( mg/ml) nd α-glucosidse ( mg/ml) were oserved. Gs chromtogrphy mss spectrometry results reveled tht the mjor components with these inhiition ilities were nd. Octnoic cid sustntilly inhiited oth α-mylse nd α-glucosidse, wheres inhiited α-glucosidse with n IC of 9.7 g/ml, which is much lower thn tht of crose ( positive control, 7.8 g/ml). I. INTRODUCTION Pper sumitted 9/1/15; revised 11/1/15; ccepted 11//15. Author for correspondence: Li-Jung Yin nd Shnn-Tzong Jing (e-mil: ljyin@mil.nkmu. edu.tw nd stjing@pu.edu.tw). 1 Deprtment of Food Science, Ntionl Tiwn Ocen University, Keelung, Tiwn, R.O.C. Deprtment of Sefood Science, Ntionl Kohsiung Mrine University, Kohsiung, Tiwn, R.O.C. 3 Nugen Bioscience (Tiwn) Co., Ltd. F, NO.35, Key Rd. Dy District, Tichung, Tiwn, R.O.C Deprtment of Food nd Nutrition, Providence University, Tichung, Tiwn, R.O.C. Morind citrifoli (noni), growing in tropicl nd sutropicl res (Lin, 13), is rich in polyphenols, flvonoids, phenolic cid (vnillic cid), vnillin, nd iridoids (sperulosidic cid nd decetylsperulosidic cid) (Dussossoy et l., 11; West et l., 11). The qulity of commercil noni fruit products vries significntly with geogrphicl conditions (Deng et l., ). Their fruits, leves, roots, stem, nd rk re frequently used in folk medicines with effectiveness in minimizing the symptoms of lifestyle-relted diseses such s hypertension, therosclerosis, stroke nd cncer in Polynesi, Thiti, Southest Asi, Austrli, nd Hwii (Mndukhil, ). Dietes is mjor metolic syndrome ffecting pproximtely million people worldwide (Hshim Hshim et l., 13) nd the numer is expected to exceed million glolly y 5 (Nguyen et l., 13). Lee et l. (1) oserved the ntidietic effect of noni fermented y Cheonggukjng on KK-Ay dietic mice. A methnol extrct of M. citrifoli reveled potentil stimultory effects on glucose uptke in 3T3-L1 dipocyte cells nd inhiitory effects on protein tyrosine phosphtse 1B (Nguyen et l., 13). The n-buoh solule phse of methnol extrct of M. citrifoli roots significntly reduced the lood glucose levels of streptozotocin (STZ)-induced dietic mice (Kmiy et l., 8). A prcticl method for mnging dietes controls postprndil hyperglycemi y inhiiting crohydrses present in the gstrointestinl trct (Ooh, 1). Cellulse plys importnt roles in the effective iologicl hydrolysis of cellulose (Perez et l., ). An extrct of noni leves hydrolyzed with cellulse from Trichoderm reesei nd Aspergillus niger reduces the glucose levels of STZ-induced dietic mice (Sugirto, 13). Lctic cid cteri (LAB) improve the intestinl microflor nd consequently reduce the risk of cncer (John et l., 7; Zhng et l., 1). In ddition, LAB increse immunity nd ntimicroil ctivity (Pwlus nd Kinghorn, 7; Gupt nd Ptel, 13) nd reduce oxidtive

2 H.-H. Lee et l.: Proiotics Fermented Noni Hydrolystes 69 stress (Wng et l., 1). Fermented noni fruits promote the growth of Lctocillus nd Bifidocterium nd regulte intrcellulr oxidtion nd inflmmtion in Cco- cells (Hung et l., 15). Moreover, fermenting noni juice y using Bifidocterium longum nd Lctocillus plntrum increses its ntioxidnt cpcity (Wng et l., 9). Most of the studies, thus fr, hve focused on identifying io-nutrients in noni, their functions nd structures, nd processes of noni fruits, roots nd leves. Studies on fcilitting the relese of ionutrients from noni fruits, roots, nd leves re scrce. In this study, cellulse hydrolysis ws used for fcilitting the relese of ionutrients from noni fruits. Furthermore, the resulting hydrolyste ws fermented using Pediococcus pentosceus BCRC 153. In ddition, the inhiition effects of relesed io-nutrients on -mylse nd -glucosidse were lso exmined. The mjor components of io-nutrients in noni extrcts were identified using gs chromtogrphy-mss spectrometry (GC-MS), nd their inhiiton effects on -mylse nd -glucosidse were further studied for elucidting the constituents inhiiting -mylse nd -glucosidse ctivities. II. MATERIALS AND MATHODS 1. Preprtion of Rw Mteril Fresh noni, purchsed from noni frm in southern Tiwn, ws homogenized using homogenizer nd used s rw mteril. Cellulse (, U/g) ws purchsed from Kwng Hw Trding Corp., Rd. Chilin, Tipei, Twin. A P. pentosceus BCRC 153 strin ws otined from the Bioresource Collection nd Reserch Center (BCRC) t the Food Industry Reserch nd Development Institute, Rd. Shih-Pin, Hsinchu, Tiwn. De Mn, Rogos, nd Shrpe (MRS) gr ws purchsed from Becton Dickinson nd Compny (Frnklin Lkes, NJ, U.S.A.). The cteril strin ws stored t - C until use nd ws ctivted through cultivtion in lctocilli MRS roth for h nd trnsferred twice efore use. Porcine pncretic -mylse (EC ), Scchromyces cerevisie -glucosidse (EC 3..1.), nd p-nitrophenyl- -Dglucopyrnoside were purchsed from Sigm-Aldrich Corp., St. Louis, Mo., U.S.A.. Preprtion of Noni Hydrolyste nd Fermented Products For fcilitting the relese of ionutrients, kg of homogenized noni (noni: wter = 1:1) using homogenizer (FD- 3S-6P, King Mech, Tipei, Tiwn), ws hydrolyzed with U/mL cellulse t C for 6 h with shking t 1 rpm. To the resulting hydrolyste, 1.5% glucose nd.6% CCO 3 were dded. After we djusted the ph to 6.5 y using C(OH), the hydrolyste ws fermented with 5% P. pentosceus BCRC 153 t 37 C for 8 h in L Biorector (MGT-SV, Micro Gint, Tichung, Tiwn). The hydrolystes nd fermented product efore freeze-dried (FD-3S-6P, King Mech, Tipei, Tiwn) t - C for dys to remove the seeds nd stored in n electronic dry cinet efore extrction. 3. Determintion of Vile Microil Counts Vile microil counts were mesured for evluting whether the noni slurry hydrolyste could e used y proiotics. Microil growth during fermenttion ws mesured using plte count method with MRS gr, fter we performed seril deciml dilutions from n initil.5 ml of smple in.5 ml of sterile.85% NCl solution. The pltes were incuted t 37 C for 8 h nd the vile proiotics were counted (Drmynti et l., 1).. Extrction of Morind Citrifoli The freeze-dried noni smple ws mixed with solvents (wter, methnol, nd ethnol) in -fold quntity nd homogenized for 3 min in refrigertor (pproximtely C). The homogenized smples were plced in hood with ventiltion t 5 C for h with shking t 1 rpm. All resulted smples were centrifuged t xg for min to remove insolule components. The vrious resulting extrcts were concentrted to minimum volume (pproximtely ml) y using rotry evportor in vcuum t C nd then freezedried. All freeze-dried extrcts were stored in n electronic dryer with < % reltive humidity efore nlyses. 5. Assys The totl phenolic content (TPC) nd inhiitory ilities of vrious freeze-dried noni extrcts ginst -mylse nd -glucosidse were mesured, nd GC-MS ssys of these extrcts were performed. 1) Totl Phenolic Content The TPC ws mesured using the Folin-Cioclteu method. To 1.5 ml of % Folin-Cioclteu phenol regent,.5 ml of smple nd 1.5 ml of 7.5% N CO 3 were dded. After 5-min rection t 5 C in the drk, sornce ws mesured t 765 nm. TPC ws clculted ccording to the stndrd curve clirted using gllic cid (Singleton et l., 1999). ) Gs Chromtogrphy Mss Spectrometry A GC-MS system, equipped with the Thermo Trce 1 gs chromtogrph, AI-13 utosmpler, nd ISQ mss spectrometer, ws used in scnning rnge of 35-. The Rxi-5MS GC column mesured m.5 mm (internl dimeter) with.1- m film thickness. The oven temperture ws incresed from C to 3 C t C/min. When the temperture reched 3 C (injector temperture),. L of extrct ws injected, nd the flow rte of helium gs ws set t 1. ml/min. 3) Qulittive nd Quntittive Anlysis y Using GC-MS The phenolic compounds of vrious extrcts were nlyzed nd identified y compring their mss spectr with those of ech reference compound in GC-MS lirries, nmely the US Ntionl Institute of Stndrds nd Technology nd Wiley th edition lirries. Quntifiction ws performed using Xcliur. softwre (Thermo Fisher Scientific, Wlthm, MA, U.S.A.).

3 6 Journl of Mrine Science nd Technology, Vol., No. 3 (16) 8 6 ph Microil counts (log CFU/mL) The Duncn multiple rnge test ws used for determining the significnce of differences within tretments. For ech tretment, 3 determintions were used, nd men vlues were clculted. P <.5 ws considered sttisticlly significnt (Norušis, 1993). III. RESULTS AND DISCUSSION Blnk F F8 Fig. 1. Chnges in ph nd microil counts of noni hydrolyste fter 8 h fermenttion y Pediococcus pentosceus BCRC 153. Blnk, un-hydrolyzed smple;, 6 h hydrolysis with U/mL cellulse; F, h fermented noni hydrolyste with 5% Pediococcus pentosceus BCRC 153; F8, 8 h fermented noni hydrolyste with 5% Pediococcus pentosceus BCRC 153. ) Inhiition Assy for -mylse The -mylse inhiitory ctivity of vrious extrcts,, nd were determined y mixing L of smple with L of 1. U/mL -mylse in mm phosphte uffer (ph 6.9 with.6 M NCl), wheres the smple in the control group ws replced with mm phosphte uffer. After -min rection t 5 C, L of.5% strch in the sme uffer ws dded. After nother -min rection t 5 C, 1. ml of dinitroslicylic regent (1% 3,5-dinitroslicylic cid nd 1% sodium potssium trtrte in. M NOH) ws dded. After 5-min rection t C (for stopping the rection), the sornce ws mesured t 5 nm (A 5 ). Inhiitory ctivity ginst -mylse (%) = [(A 5 (control) A 5 (smple)]/a 5 (control) % (Kim et l., 5; Dong et l., 1). The IC ws defined s the inhiitor concentrtion required for inhiiting % of - mylse ctivity. 5) Inhiition Assy for -glucosidse The -glucosidse inhiitory ctivity of vrious extrcts,, nd ws determined y mixing L of smple with L of. U/mL -glucosidse in.1 M phosphte uffer (ph 6.8), wheres the smple in the control group ws replced with.1 M phosphte uffer. After -min rection t 37 C, L of 5 mm p-nitrophenyl- -Dglucopyrnoside in the sme uffer (ph 6.8) ws dded. After nother -min rection t 37 C, the rection ws terminted using 1 L of. M N CO 3. The sornce ws mesured t 5 nm (A 5 ). Inhiitory ctivity ginst -glucosidse (%) = [(A 5 (control) A 5 (smple)]/a 5 (control) % (Andrde-Cetto et l., 8; Kwon et l., 8). The IC ws defined s the inhiitor concentrtion required for inhiiting % of -glucosidse ctivity. 6) Sttisticl Anlysis 1. ph nd Microil Counts After 6 h of hydrolysis with U/mL cellulse t C, the ph slightly decresed from.71 to.6 (Fig. 1). We previously tested severl proiotics, ut none of them could grow in the noni slurry nd its cellulse-hydrolyzed smples, except for P. pentosceus BCRC 153, which grew in noni hydrolyste with.6% CCO 3 (Lee et l., 15). Therefore,.6% CCO 3 ws dded to the resulting hydrolyste. After 8 h of fermenttion with P. pentosceus BCRC 153 t 37 C, LAB vile counts incresed to 9.17 log CFU/mL, nd the ph decresed to 5.7 (Fig. 1). This increse in LAB counts nd the decrese in ph suggested tht the noni hydrolyste could e used y P. pentosceus. Although glucose is the min crohydrte derived from cellulse hydrolysis nd cn e used y proiotics (Kimoto-Nir et l., ; Hernndez- Hernndez et l., 1), the noni hydrolyste nd commercil noni juice with no crontes could not e used y the proiotics tested (Lee et l., 15). Adding CCO 3 in the noni hydrolyste ws essentil for successful LAB fermenttion. MRS gr with.1% CCO 3 sustntilly improved proiotic growth during dongchimi kimchi fermenttion ecuse of the uffering effect (Che et l., 9). Therefore, during LAB fermenttion, proiotic growth is inhiited ecuse of lctic cid. Undissocited lctic cid psses through the cteril cytoplsmic memrne nd dissocites inside the cell, cusing cytoplsmic cidifiction nd proton motive force filure, which consequently reduces the mount of energy required for cell growth (Mrkovic et l., 11).. Effects of Hydrolysis nd Fermenttion on the Extrction Yield of Noni Unhydrolyzed, cellulse-hydrolyzed, nd dditionlly fermented noni were extrcted using wter, methnol, nd ethnol. The yields of the wter extrcts of unhydrolyzed, hydrolyzed, nd fermented products were 13.7, 7.8, nd mg/g, respectively (in dry se), while those of methnol nd ethnol extrcts were 1.3, 36.7, nd 15.8 mg/g nd 87.8, 1., nd mg/g, respectively (Tle 1). Regrdless of the solvent used for extrction, the extrction yields of hydrolyzed smples nd fermented hydrolystes were much higher thn those of unhydrolyzed smples (Tle 1). Cellulse plys mjor role in the effective iologicl hydrolysis of cellulose into glucose through the synergistic ctions of endo- -1,-glucnse (EC 3..1.), celloiohydrolse (EC ), nd -D-glucosidse (EC ) (Perez et l., ). The ction mode of cellulses on polymers is either exoclevge or endoclevge, nd ll cellulses trget the specific

4 H.-H. Lee et l.: Proiotics Fermented Noni Hydrolystes 651 Tle 1. Extrction yield of Morind citrifoli (noni) extrcts. Extrction yield (mg/g) Extrct solvent Blnk * F8 Wter ** 7.8.7c Methnol c Ethnol c * Blnk: un-hydrolyzed smple; : 6 h hydrolysis with U/mL cellulse; F8: 8 h fermented noni hydrolyste with 5% Pediococcus pentosceus BCRC 153. ** ~c: Vlues with different letters t the sme row re differ significntly (P <.5). Totl phenol content (Reltive gllic cid mg/g dried smple) 8 6 c wter c methnol Solvent of extrction Blnk F8 c ethnol Fig.. Effects of the hydrolysis nd fermenttion on totl phenolic content of noni. Blnk, nd F8 refer to the footnote of Fig. 1. ~c: mens with different extrcts within the sme solvent extrction re significntly different (P <.5). clevge of -1,-glycosidic onds (Wood nd McCre, 1979). The higher extrction yields of hydrolyzed smples nd fermented hydrolystes thn those of unhydrolyzed smples indicted tht cell wll lysis occurs in noni fruits during cellulse hydrolysis, fcilitting the relese of these extrcts. 3. Effects of Hydrolysis nd Fermenttion on Totl Phenolic Content of Noni The TPCs of wter extrcts of unhydrolyzed, hydrolyzed, nd fermented smples were., 7.5, nd 6.71 mg/g (in dry se), respectively, while those of methnol nd ethnol extrcts were 3.5, 6.3, 5.8 mg/g nd.3, 3.18, nd 3.3 mg/g, respectively (Fig. ). Both hydrolysis nd fermenttion significntly incresed the TPCs of the extrcts. Although the TPC of methnol extrct of noni otined y Krishnih et l. (15) ws higher thn the TPC otined in this study (.3 mg/g vs. 3.5 mg/g), this might e due to the differences in hrvest re nd seson. In the present study, significnt increses in the TPCs of vrious extrction solvents fter cellulse hydrolysis showed tht cell wll disruption occurred during hydrolysis, relesing most ionutrients. By contrst, slight decreses in the TPCs might e ttriuted to the utiliztion y LAB (Fig. ). Fig. 3. IC vlues of lph-mylse inhiition (mg dried smple / ml) IC vlues of lph-glucosidse inhiition (mg dried smple / ml) wter c methnol Solvent of extrction () wter methnol Solvent of extrction () c c ethnol Blnk F8 Blnk F8 ethnol Effects of hydrolysis nd fermenttion of noni on the inhiition () α-mylse, () α-glucosidse. Blnk, nd F8 refer to the footnote of Fig. 1. ~c: mens with different extrcts within the sme solvent extrction re significntly different (P <.5).. Effects of Hydrolysis nd Fermenttion on -mylse nd -glucosidse Inhiition of Noni For -mylse, the IC of the wter extrcts of unhydrolyzed, hydrolyzed, nd fermented products were.8,.6, nd.35 mg/ml, respectively, while those of methnol nd ethnol extrcts were.3, 1., nd 1.61 mg/ml nd 18.53, 5.88, nd.57 mg/ml, respectively (Fig. 3). For -glucosidse, the IC of the wter extrcts of unhydrolyzed, hydrolyzed, nd fermented products were 6.7, 9.7, nd 11.9 mg/ml, respectively, while those of methnol nd ethnol extrcts were 16.65, 6.9, nd 3.73 mg/ml nd 9.55, 5.6, nd 9.7 mg/ml, respectively (Fig. 3). The iologicl functionl effects of polyphenols in fruits nd vegetles fruits hve long een studied. These compounds hve inhiitory ility ginst crohydrte hydrolyzing enzymes (Mi et l., 7; Cheplick et l., ; Rnill et l., ; Wongs nd Zmludien, 1). By using GC-MS, nd were identified s mjor ionutrients in ll of the noni extrcts (Figs. nd 5). The IC vlues of were.9 nd.9 mg/ml for inhiiting α-mylse nd -glucosidse ctivities,

5 65 Journl of Mrine Science nd Technology, Vol., No. 3 (16) () stndrd: 7 (e) stndrd: () wter extrct: 7 (f) wter extrct: (c) methnol extrct: 7 7 (g) methnol extrct: (d) ethnol extrct: 7 7 (h) ethnol extrct: Fig.. GC-MS chromtogrms with qulittive of noni extrct on nd. respectively (Tle ). Scopoletin could not inhiit -mylse ctivity, ut considerly inhiited -glucosidse ctivity. The IC of ws 9.7 g/ml, which ws considerly lower thn tht of the positive control crose (7.8 g/ml), n nti-dietic drug (Tle ). This result showed tht the inhiitory ility of ws lmost -fold higher thn tht of crose. Scopoletin lso showed ntihyperglycemic ctivity in STZ-induced dietic rts (Verm et l., 13). The nd contents of vrious extrcts significntly incresed fter cellulse hydrolysis nd dditionl LAB fermenttion (Fig. 6). A slight decrese in content fter LAB fermenttion ws oserved (Fig. 6) nd my e ttriuted to phenol consumption y LAB. Othmn et l. (8) reported tht proiotics use totl phenols during fermenttion. According to the dt otined, considerle mounts of nd were relesed fter cellulse hydrolysis nd dditionl LAB fermenttion

6 H.-H. Lee et l.: Proiotics Fermented Noni Hydrolystes 653 Fig () wter extrct: lnk () wter extrct: (c) wter extrct: F (d) methnol extrct: lnk (e) methnol extrct: (f) methnol extrct: F (g) ethnol extrct: lnk (h) ethnol extrct: (i) ethnol extrct: F8 GC-MS chromtogrms of vrious extrcts of noni with fold dilution. Mjor chnges were identified s nd. () wter extrct-lnk, () wter extrct-, (c) wter extrct-f8, (d) methnol extrct-lnk, (e) methnol extrct-, (f) methnol extrct-f8, (g) ethnol extrct-lnk, (h) ethnol extrct-, (i) ethnol extrct-f8. Blnk, un-hydrolyzed smple;, 6 h hydrolysis with U/mL cellulse; F8, 8 h fermented noni hydrolyste with 5% Pediococcus pentosceus BCRC 153. Tle. Inhiitory ctivity (IC ) of nd octnoic cid on -mylse nd -glucosidse. Compounds -Amylse IC (mg/ml) -Glucosidse Acrose * Scopoletin ND **.97. Octnoic cid * Acrose, positive control. ** ND, not detected. (Fig. 6). Becuse of their low IC vlues, hydrolyzed nd dditionlly fermented noni fruits my replce crose. 5. Qulittive nd Quntittive Anlysis of Vrious Noni Extrcts y Using GC-MS Two components, nd, of vrious noni extrcts (wter, methnol, nd ethnol extrcts) were identified nd quntified using GC-MS (Figs. nd 5). Fig. 6 shows the mounts of nd fter the quntittive results of GC-MS were clculted. The octnoic cid contents of wter extrcts of unhydrolyzed, hydrolyzed, nd fermented products were 1.7, 6.7, nd 7.59 mg/g (in dry se), respectively, nd those of methnol nd ethnol extrcts were.5, 6.71, nd 7. mg/g nd 3.7,.1, nd 5.15 mg/g Octnoic cid content (mg/g dry weight) Scopoletin content (mg/g dry weight) wter wter methnol Solvent of extrction () methnol Solvent of extrction () ethnol ethnol Blnk F8 Blnk F8 Fig. 6. Quntittive ingredients in vrious extrcts of noni smples (), (). Blnk, nd F8 refer to the footnote of Fig. 1.

7 65 Journl of Mrine Science nd Technology, Vol., No. 3 (16) (in dry se), respectively (Fig. 6()). Pino et l. (9) used diethyl ether/pentne (:1 v/v) for extrcting nd otined t pproximtely 3.6 mg/g (in dry se) from edile noni fruits. However, in this study, the content of vrious extrcts fter cellulse hydrolysis or LAB fermenttion ws lmost fold higher thn tht otined y Pino et l. (9). The contents of wter extrcts of unhydrolyzed, hydrolyzed, nd fermented products were.9, 6., nd 3.97 mg/g (in dry se), respectively, while those of methnol nd ethnol extrcts of unhydrolyzed, hydrolyzed, nd fermented products were 3.33, 5.8, nd.7 mg/g nd.9, 3.83, nd 3.6 mg/g (in dry se), respectively (Fig. 6()). The content of vrious extrcts in this study ws much higher thn tht of oiled wter extrct of noni powder [. g/g (in dry se)] otined y Prpitrkool nd Ithrt () (Fig. 6()). However, Pndy et l. (1) otined higher content (18.9 mg/g) in methnol extrct of noni thn we did. This difference my e ttriuted to differences in the extrction time ( h vs. h) nd species. CONCLUSION The present study results reveled tht cellulse hydrolysis nd P. pentosceus fermenttion fcilitted ionutrient relese from noni fruits. Increses in extrction yields nd TPCs were otined fter hydrolysis nd LAB fermenttion, consequently incresing the inhiitory ilities ginst α-mylse nd - glucosidse. The mjor components in ionutrients tht cused -mylse nd -glucosidse inhiition were nd. The otined dt suggest tht cellulse hydrolysis nd dditionlly fermented noni hydrolystes cn e processed into nutritionl supplements with ntihypoglycemic properties. ACKNOWLEDGMENTS This study ws supported y grnt (NSC-1-E-- 13) from the Ministry of Science nd Technology, Tiwn. REFERENCES Andrde-Cetto, A., J. Becerr-Jimenez nd R. Crdens-Vzquez (8). Alfglucosidse-inhiiting ctivity of some mexicn plnts used in the tretment of type dietes. Journl of Ethnophrmcology 116, 7-3. Che, S., J. Moon, J. Jung, J. Kim, H. Eom, S. 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