Comparative Biochemistry and Physiology Part C 130 Ž2001. 369᎐377
Protective effects of Panax ginseng on muscle injury and inflammation after eccentric exercise Antonio C. Cabral de Oliveira, Andrea C. Perez, Gracia Merino, Julio G. Prieto, Ana I. AlvarezU Department of Physiology, Uni¨ ersity of Leon, Leon 24071, Spain Received 3 June 2001; received in revised form 6 August 2001; accepted 22 August 2001
Abstract Eccentric muscle contraction causes fibre injury associated with disruption of the myofibrillar cytoskeleton. The medicinal plant Panax ginseng C.A. Meyer, known for its therapeutic properties, was studied to explore its protective effects after eccentric contraction. A crude extract and a standardised extract ŽG115. of different saponin compositions were tested as to their efficacy in reducing lipid peroxidation, inflammation and release of myocellular proteins after the realisation of an eccentric contraction protocol on a rat treadmill. Plasma creatine kinase ŽCK. levels were significantly reduced by approximately 25% after ingestion of both extracts of ginseng. Both extracts reduced lipid peroxidation by approximately 15% as measured by malondialdehyde levels. -Glucuronidase concentrations and glucose-6-phosphate dehydrogenase ŽG6PDH. levels, which can be considered markers of inflammation, were also significantly reduced. The values of -glucuronidase were increased from 35.9" 1.5 to 128.4" 8.1 in vastus and to 131.1" 12.1 U gy1 in rectus, the protection due to ginseng administration being approximately 40% in both muscles. Both extracts appeared to be equally effective in reducing injuries and inflammation caused by eccentric muscle contractions. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Eccentric exercise; Ginsenosides; Inflammation; Muscle injury; Panax ginseng; Creatine kinase; Malondialdehyde; -Glucuronidase; Glucose-6-phosphate dehydrogenase
1. Introduction Ginseng, the root of the Araliaceous plant, is traditionally used in Chinese medicine. It is considered to have salutary effects and stimulant actions on physical capacity ŽBahrke and Morgan, 2000.. The composition of ginseng is complex and many of its compounds exhibit pharmacological U
Corresponding author. Departmento Fisiologia, Facultad de Veterinaria, Universidad de Leon, Leon 24071, Spain. Tel.: q34-987-291266; fax: q34-987-291267. E-mail address:
[email protected] ŽA.I. Alvarez..
actions. The major active components of ginseng are ginsenosides, although compounds other than these may also have biological activity. The ginsenosides are a diverse group of steroidal saponins showing the ability to target a vast range of tissues. The two major groups of ginsenosides are the Rb and Rg groups, which have the 20 ŽS. protopanaxadiol and 20 ŽS. protopanaxatriol groups, respectively, as the sapogenins. The Rb group includes the ginsenosides Rb1 , Rb 2 , Rc and Rd, while the Rg group includes Re, Rf and Rg 1 as the main compounds. However, many of the mechanisms responsible
1532-0456r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 1 . 0 0 2 6 2 - 9
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for ginsenoside activity remain unknown. The actions of ginseng actions involve anti-aging, immunoenhancement, anti-tumour, anti-stress, antioxidant and organ protective effects ŽMatsuda et al., 1990; Gillis, 1997; Attele et al., 1999.. Some of the individual effects of ginsenosides have been studied; thus, Rb1 increases thermogenesis and cold tolerance ŽWang and Lee, 2000.; restores the action potentials of free radical-damaged cells to normal indicating its anti-oxidative action, and shows calcium channel blockade activity, like Rb 2 or Rb 3 ŽJiang et al., 1992., and has similar effects to those of Rg 1 in pulmonary vessel relaxation, probably mediated by a NO release action ŽChen, 1996.. Three species with medicinal properties are currently recognised: Panax ginseng ŽKorean ginseng., Panax quinquefolius ŽAmerican ginseng. and Panax japonicus ŽJapanese ginseng.. The actual composition of ginseng preparations has been a persistent problem and the observed differences in their effects may be due to the dosage, form of administration and type of ginseng employed. Even the potency of extracts may differ when the plants are cultivated in different areas ŽAttele et al., 1999.. There may also be interactions with diet, life style, exercise and other drugs. Although such effects are known world-wide, scientific proof remains lacking. Reports are often contradictory, perhaps because the ginsenoside content can differ. Therefore, the use of standardised, authentic root both in research and by the public is to be advocated. The aim of the present study was to compare the effects of two preparations of Panax ginseng C.A. Meyer on muscle damage and inflammatory response induced by eccentric contraction. The first was a crude extract of Panax ginseng or Red Korean ginseng ŽGred. and the second was a standardised preparation containing 13 ginsenosides, currently marketed and known as G115. Excessive physical exercise, such as eccentric exercise, causes injuries to the skeletal muscle fibres of humans and animals unaccustomed to such exercise. Eccentric contraction generates high forces per active cross-sectional area; the initial lesions are subcellular and segmental and mainly occur in a proportion of fibres in an affected skeletal muscle. The early changes in exercised-induced muscle injury consist of a disruption in the myofibrillar banding pattern and disruption of the sarcolemma. From 2 to 6 h later,
secondary changes such as fibre autophagy and heterophagy by macrophages are observable in the muscle fibres. This stage continues for the next 2᎐4 days. Regenerative processes usually start within 4᎐6 days ŽSorichter et al., 1999.. The eccentric running protocol described by Armstrong et al. Ž1983. was the non-invasive method used in this study. The amount of muscle injury after exercise was measured biochemically ŽSalminen and Kihlstrom, ¨ 1985; Clarkson et al., 1986. by analysing the activities of the following: creatine kinase ŽCK. in plasma as a reliable quantitative indicator of membrane integrity, and muscle -glucuronidase and glucose-6-phosphate dehydrogenase activities, used as indicators of inflammation and the damage generated by free radicals after eccentric exercise. The increased vulnerability of the muscle membrane was measured by lipid peroxidation ŽMDA..
2. Methods 2.1. Drugs and chemicals The standardised Panax ginseng extract G115 was a generous gift from Pharmaton S.A., Lugano ŽSwitzerland. and the crude extract was from Korhispana SA Barcelona ŽSpain.. Standard enzymes or products were obtained from Sigma Chemical Company ŽSpain.. 2.2. Ginsenoside quantification Extraction of ginsenosides was based on the method described by Samukawa et al. Ž1995.. Freeze-dried powdered samples Ž50 mg. were washed in petroleum ether Ž3 = 8 ml. and centrifuged at 33 520 = g for 10 min. The pellet was extracted and sonicated for 15 min with 15 ml of 70% methanol and centrifuged again at 33 520 = g for 10 min. After evaporating the methanolic extract to dryness and dissolving the residue in 10 ml of acetonitrile:water Ž80:20., 20 l of the ginsenoside extract was loaded onto a HewlettPackard Lichrospher 䊛 100 Diol Ž5 m. column Ž250 = 4 mm. connected to a Pharmacia LKB h.p.l.c. system. The solvent mixture of acetonitrile᎐water Ž4.5:1 v vy1 , pH 2.5 with H 3 PO4 . was eluted at a flow rate of 0.8 ml miny1 and ginsenosides were detected at 202 nm. The ginsenoside
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contents of the samples analysed were expressed as mg ginsenosides gy1 dry wt. 2.3. Animals and exercise protocol Male Wistar rats weighing 200 " 30 g obtained from IFFA Credo ŽMadrid, Spain. were used. All animal handling practices complied with the principles of Council Directive 86r609r of the European Community concerning the ‘Protection of Animal used for Experimental an other Scientific Purposes’. The animals Ž n s 6 for each group. were divided as follows: two groups of animals received daily 1 ml of ginseng extract G115 or crude extract ŽGred., respectively, at a dose of 100 mg kgy1 dayy1 along the 3 months of the experimental period by gastric intubation and were made to perform the exercise protocol described below. This dose was chosen based on our previous works ŽVoces et al., 1999.. One group received 1 ml of saline solution per day by gastric intubation and did not perform exercise Žsedentary. and another group ᎏ called control exercised ᎏ received saline and performed exercise on a rodent treadmill with the following protocol described by Armstrong et al. Ž1983.. The rats ran an intermittent protocol downhill Žy16⬚ incline. at 16 m miny1 for a total of 90 min; 5-min bouts Ž18 bouts. separated by 2-min rests. At the time of killing the animals were anaesthetised with pentobarbital sodium Ž5 mg 100 gy1 body wt. i.p.., and the muscles rapidly excised, trimmed of extraneous fat and connective tissue, immersed in isopentane, dropped into liquid nitrogen and stored at y70⬚ until further analysis. Tissues were homogenised at a proportion of 1:10 wrvy1 in a phosphate buffer 0.1 M ŽpH 7.4. at 0⬚C and centrifuged at 750 = g in order to separate the cell residues. The muscles studied were the soleus, rectus, extensor digitorum longus ŽEDL., tibialis anterior and vastus intermedius. Blood was sampled from the tail vein and used to determine plasma CK levels. 2.4. Biochemical analyses Plasma CK levels were analysed using a commercially available kit ŽSigma Chemical., immediately after Ž0 h. or in different times Ž24, 48, 72, 96, 120, 144 and 168 h. after eccentric exercise.
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2.5. Malondialdehyde (MDA) measurements Malondialdehyde ŽMDA. was assayed by quantifying the release of malonyldialdehyde ŽMDA. in muscle homogenates using the 2-thiobarbituric acid ŽTBA. ᎐trichloroacetic acid ŽTCA. ᎐HCl reagent, following the method of Buege and Aust Ž1978.. An aliquot of the homogenate and 1 ml of reagent were added to the centrifuge tubes. The mixture was kept at 100⬚C. After a short centrifugation at 3000 = g for 10 min, the absorbance of the supernatant was measured by spectrophotometry at 535 nm. Parallel standards containing tetramethoxypropane 5 M ŽTMP . were processed. 2.6. Muscle G-6-phosphate dehydrogenase analyses G6PDH was analysed by spectrophotometry following the reduction of NADPq, according to Cohen and Rosemeyer Ž1975.. Samples of homogenate Ž100 l. were added to 0.8 M Tris᎐HCl, pH 9.0, NADP Ž1 mM., and glucose 6-phosphate Ž10 mM. at a final volume of 1 ml. The rate of exchange in absorbance was measured at 340 nm. Enzymatic activity was defined as the quantity of enzyme that reduces 1 mol of NADPH miny1 at 25⬚C per g wt. of wet tissue. 2.7. -Glucuronidase -Glucuronidase was assayed according to Rosenfeld et al. Ž1983. measuring the hydrolysis of p-nitrophenyl--D-glucuronide. The homogenate Ž200 l. was incubated for 60 min at 37⬚C at a final volume of 1 ml containing 0.2 M sodium acetate buffer, pH 4.5, 0.1% bovine serum albumin, and 10 mM p-nitrophenyl-- D glucuronide. The reaction was stopped by adding 2.0 ml of 0.1 M NaOH and 3 ml of water, and absorbance was measured at 400 nm. One unit is the activity that catalyses the release of 1 mol of p-nitrophenol miny1 from the substrate. Taking into account that the development of damage associated with eccentric contraction does not occur immediately after exercise and that it shows temporal variability, the parameters used in the present study to calculate muscle damage were determined immediately after eccentric exercise and over 1 week after this.
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Table 1 Main composition of ginsenosides Žmg gy1 dry wt.. from two types of ginseng extract: standardized ginseng G115 and a crude extract of red ginseng ŽGred. analysed by h.p.l.c.
G115 Gred
Rb1
Rb2
Rc
Rd
Re
Rf
Rg1
1.39" 0.12 10.50" 1.05
11.59" 1.30 10.24" 0.98
3.99" 0.20 9.93" 0.97
9.06" 1.05 5.02" 0.49
9.59" 0.85 5.05" 0.51
2.36" 0.25 1.71" 0.18
4.61" 0.43 2.78" 0.28
2.8. Statistical analysis The results are expressed as means " S.E. mean. The statistical significance of differences between the groups was determined by ANOVA followed by a post-hoc Newman᎐Keuls test. A probability Ž P . value of less than 0.05 was taken to indicate statistical significance.
3. Results The compositions of the ginsenosides are shown in Table 1. Differences were found in ginsenosides Rb1 and Rg 1 , which according to a few authors are the most efficient compounds ŽTanaka and Kasai, 1984., although other ginsenoside components also showed smaller differences. Fig. 1 shows a time course of plasma CK in sedentary and exercised animals. Immediately following exercise Ž0 h. a significant increase in plasma CK activity was observed. Plasma CK levels were analysed over a very long period of time: up to 168 h after the performance of eccen-
tric exercise, when they almost returned to basal values. The highest levels of CK activity were noted 96 h after exercise Ž523 " 35 U ly1 ., and the ginseng-treated groups showed significantly Ž P- 0.05. lower values when compared with their control Ž381 " 26 U ly1 and 396 " 45 U ly1 for G115 and Gred, respectively.. These levels were also significantly Ž P - 0.05. lower in both ginseng-treated groups after 120 h of exercise. Muscle -glucuronidase and G6PDH activities in the five muscles are shown in Figs. 2 and 3, respectively. The exercised animals showed an increase in these enzyme activities after the performance of the exercise protocol. The results revealed that there were always significant differences Ž P- 0.05. between the sedentary and control exercised animals. The muscles most affected were the vastus, soleus and rectus. Changes in enzyme activities occurring in any of the five muscles were seen in the 72᎐96-h period after exercise, except for the vastus, in which they were observed a few hours earlier Ž48 h. in the case of G6PDH. Regarding the vastus, the values were significantly Ž P- 0.05. increased from 0.30" 0.01
Fig. 1. Time course of plasma creatine kinase ŽCK; U ly1 . in sedentary and exercised animals immediately after Ž0 h. and at different times after eccentric exercises. Open bars represent G115 treated-animals, and hatched bars Gred-treated animals. Values are means " S.E. of 4᎐6 animals per group and per period of time. U P- 0.05 of treated vs. control exercised animals.
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Fig. 2. Effect of ginseng extract ŽG115. and red Korean ginseng ŽGred. administration Ž100 mg kgy1 . to rats subjected to eccentric exercise at different periods of time, showing -glucuronidase concentrations ŽU gy1 miny1 .. Values are means " S.E. of 6 animals per group. U P- 0.05 of treated vs. control exercised animals. 噛 P- 0.05 of sedentary vs. control exercised animals.
mol miny1 gy1 in sedentary to 0.88" 0.03 mol miny1 gy1 in control animals. The treated animals showed a significant Ž P- 0.05. decrease in enzyme activity to 0.57" 0.08 mol miny1 gy1 and 0.70" 0.03 mol miny1 gy1 for G115 and Gred, respectively. -Glucuronidase activity was increased Ž P- 0.05. by over 200% on comparing the rectus and vastus in the sedentary and control
animals, while in the ginseng-treated groups a significant Ž P- 0.05. reduction of approximately 40% in enzyme activity was found. The tibial and EDL muscles showed the lowest increments in these enzyme activities, and in a similar manner were significantly Ž P- 0.05. reduced by either ginseng extract administration. Fig. 4 shows the levels of MDA after eccentric
Fig. 3. Effect of ginseng extract ŽG115. and red Korean ginseng ŽGred. administration Ž100 mg kgy1 . to rats subjected to eccentric exercise at different periods of time, showing glucose-6-phosphate dehydrogenase ŽG6PDH. concentrations Žmol gy1 miny1 .. Values are means " S.E. of 6 animals per group. U P- 0.05 of treated vs. control exercised animals. 噛 P- 0.05 of sedentary vs. control exercised animals.
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Fig. 4. Effect of ginseng extract ŽG115. and red Korean ginseng ŽGred. administration Ž100 mg kgy1 . to rats subjected to eccentric exercise at different periods of time, showing malondialdehyde ŽMDA. levels Žnmol gy1 muscle.. Values are means " S.E. of 6 animals per group. U P- 0.05 of treated vs. control exercised animals. 噛 P- 0.05 of sedentary vs. control exercised animals.
exercise. High MDA levels, as a sign of membrane disruption, were noted a few hours after the exercise. The exercised animals had significantly higher Ž P- 0.05. MDA levels as compared to the sedentary ones. The largest differences between the sedentary and control Žexercised. rats were seen in the muscles with the highest percentage of glycolytic fibres Žless oxidative potential ., i.e. rectus, EDL and tibial muscle. However, with both ginseng treatments MDA concentrations were significantly Ž P- 0.05. reduced in all the muscles studied over the same period of time stated for the muscle enzymes above.
4. Discussion Eccentric exercise can cause muscle damage and the development of this injury appears to involve a complex network of different mechanisms: first when the sarcomeres are forced to stretch to a greater extent: second through a potential mechanism involving the loss of calcium homeostasis, in which membrane damage leads to increased intracellular calcium, and finally a cellular stress response, including activation of inflammatory cells and, as suggested in recent stud-
ies, the involvement of nitric oxide ŽReid, 1998; Sorichter et al., 1999.. Lipid peroxidation and the increased levels of proteins, such as CK, in the circulation as a response to myofibrilar disintegration were significantly reduced by ginseng administration ŽFig. 1.. The presence in the blood of enzymes that are normally localised in muscle fibres is taken as evidence of disruption or increased permeability of the muscle cell membranes, at this respect the CK increase in plasma is considered one of the best indicator of muscle damage in eccentric contraction ŽArmstrong et al., 1983.. The majority of authors have found a mismatch between the time-courses of the damage with delayed peak enzyme efflux occurring 4᎐7 days post-exercise ŽCleak and Eston, 1992.. Ginseng had a protective role, decreasing such elevated CK levels significantly, although it did not prevent leakage of CK from the muscle into the blood after exercise. -Glucuronidase activity, which correlates well with the overall histological changes in injured muscle, showed decreased activity in the soleus, rectus, EDL, tibial and vastus muscles after ginseng administration Žboth types; Fig. 2.. The highest protection was found in the vastus in the early stage of the inflammatory process Ž72 h..
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Leukocytes have high specific activities of G6PDH, which provide a simple means of quantifying inflammatory or degenerative processes. G6PDH was elevated in the same approximate time period as -glucuronidase, which in turn is associated with macrophage activity and degeneration of fibres, which was also protected by ginseng administration. The increases in the activity of both these enzymes are thought to partly reflect muscle damage in the same way: the higher the number of inflammatory cells, the larger the number of injured fibres. In addition, total -glucuronidase activity reflects overall muscle damage because it also accounts for the increased activity originating from the affected but surviving muscle fibres ŽSalminen, 1985.. Salminen and Kihlstrom ¨ Ž1985. showed that -glucuronidase activity correlates significantly with the amount of histopathological changes. The highest protection by both ginseng extracts was observed in the most damaged muscles Žrectus and vastus., which are extensor muscles composed mainly of type I fibres. Injuries in the hind limbs have been observed more frequently in the extensor than the flexor muscle after prolonged exercise ŽKomulainen and Vihko, 1998.. In the extensor muscle groups, slow oxidative fibres are predominant in the deep portion ŽVihko et al., 1978.. The degree of exercise-induced damage varies in different muscles, and is probably associated with the different recruitment of muscles during running and possibly with their anatomical location in separate compartments ŽSalminen and Vihko, 1983.. This is in agreement with the findings of Vihko et al. Ž1978., who found the largest increases in hydrolases to occur mainly in oxidative fibres when mice were subjected to exhaustive running. Several other rodent studies have also demonstrated this selective damage between different fibre types, suggesting that oxidative fibres are more susceptible to exercise-induced damage than glycolytic fibres ŽArmstrong et al., 1983.. In humans, exercise-induced pathological changes have been shown to be restricted mainly to type II fibres ŽFriden ´ et al., 1983.. The protective effects of ginseng were mainly seen in the 72᎐96-h period after the bout of exercise, depending on the muscle in question, and this correlates well with the alteration of MDA levels also seen in the 72᎐96-h period, with the exception of
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the vastus Žwhich occurred at 48 h., after eccentric exercise ŽFig. 4.. It is well known that different ginsenosides exert different pharmacological effects. Among all the ginsenosides Rb1 and Rg 1 could be the most effective compounds found in ginseng ŽTanaka and Kasai, 1984.. The composition of Rb1 is different in the two extracts ŽTable 1.. Rb1 has been reported to be an inhibitor of Ca2qcalmodulin protein kinase, eliciting a disturbance in Ca2q concentrations and inhibiting Ca2q accumulation in liver homogenates or microsomes and inhibiting protein phosphorylation, such as glycogen synthase ŽPark et al., 1996.. Glycogen synthase is inactive in the synthesis of glycogen, an important fuel for muscle metabolism, when it is phosphorylated in the liver. Williams and Klug Ž1995. argued that control of intracellular Ca2q concentration is a critical link between muscle activity and fatigue. Additionally, Chin and Allen Ž1997. demonstrated that depletion of muscle glycogen also modifies Ca2q release, reduces contractile performance, and leads to muscle fatigue. The concomitant reduction in the maintenance of energy use may be an important step in maintaining the structural and functional integrity of the cell. Regarding membrane integrity, many studies have already reported the ability of ginsenosides to target cell membranes and that they also freely traverse the membrane, modify its physical properties, interact directly with membrane proteins, and even become incorporated into membranes ŽAttele et al., 1999.. The muscles that showed the highest increases in MDA levels were the EDL and tibial muscle. These muscles are mainly composed of type IIb fibres and show the lowest antioxidant capacity ŽJi et al., 1992.. Ginseng extracts exhibit antioxidant capacity. According to Jiang et al. Ž1992., the degree of antioxidant action shown by the main saponins is Rb1 ) Rb 2 ) Rb 3 . By contrast, analysing Vietnamese ginseng, Huong et al. Ž1998. concluded that it is the minor components contained in this species, and not the main saponin, that are responsible for the inhibition of MDA formation Žbrain and liver.. A possible mechanism that could be invoked to explain the suppression of lipid peroxidation is through the inhibition of enzymes such as lipoxygenase involved in the metabolism of unsaturated
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fatty acids, arachidonic acid, to prostaglandins. Ginseng has well known anti-inflammatory actions ŽMatsuda et al., 1990. and such effects could be caused by a decrease in phospholipase A 2 activity ŽLi and Chu, 1999. and by the reinforcement of muscle fibre membranes in a similar manner to another anti-inflammatory agent, i.e. prednisone ŽJacobs et al., 1996.. Several authors have found that decreased phospholipase A 2 activity can lead to a decrease in the hydrolysis of membrane phospholipids, decreased membrane fluidity, and a decreased Ca2q influx and muscle damage ŽFlower, 1990; Jacobs et al., 1996.. Free radicals may be generated by several pathways after eccentric exercise in skeletal muscle and many reports have indicated that they may play a role in muscle damage unless antioxidant enzymes do not respond adaptively by modifying their activities in tissues ŽSchauer et al., 1990.. The Rb 2 compositions of the two extracts were basically identical and this saponin is a specific inducer of the Cu,Zn-superoxide dismutase ŽSOD1. gene, which is one of the major antioxidant enzymes ŽKim et al., 1996.. The effects of Panax ginseng may develop very rapidly, in this case after 3 months of ginseng administration. Although both ginsengs differ in their contents, their efficacy in reducing muscle damage seems to be similar since they reduced plasma CK, MDA, G6PDH and -glucuronidase levels in all the muscles studied. This means that membrane integrity is maintained and muscle inflammation reduced. In conclusion, both extracts of ginseng protected the muscles studied from injury and inflammation after eccentric exercise, although the exact mechanism of this protection needs to be further evaluated.
Acknowledgements The authors thank Pharmaton S.A. and Korhispana S.A. for the Ginseng extracts. ACP is supported by a fellowship from the Comision Interministerial de Ciencia y Tecnologia de Espana. References Armstrong, R.B., Ogilvie, R.W., Schwane, J.A., 1983. Eccentric exercise-induced injury to rat skeletal muscle. J. Appl. Physiol. 54, 80᎐93.
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