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Anti-inflammatory Effect of Traditional Herbal Formula Jonlon-5 Decoction on Carrageenan-induced Acute Paw Edema in Rats

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Published: 18th May 2020 in Sciences

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Anti-inflammatory effect of traditional herbal formula Jonlon-5 decoction on carrageenan-induced acute paw edema in rats

Davaadulam Pj*,1,+,Uuganbayar B2 & Oyuntsesteg N1

1School of Traditional Medicine, Mongolian National University of Medical Sciences, Zorigstreet, Ulaanbaatar-210648, Mongolia

2Institute of Traditional Medicine and Technology of Mongolia Khan Uul district, Chinggis Avenue, Ulaanbaatar 17032, Mongolia

E-mail: +davaadulam.pj@gmail.com

This study aimed to assess the anti-inflammatory effects of a traditional Mongolian medicine-Jonlon-5 against inflammation in an animal model. We also evaluated the acute and sub-acute toxicity of Jonlon-5. The level of acute toxicity of Jonlon-5 was determined in accordance with the Organization for Economic Co-operation and Development guidelines. A sub-acute toxicity study was done by oral administration of Jonlon-5 with daily doses of 100, 200, and 500 mg/kg body weight for 28 days. Paw edema was produced by a sub plantar infection of 1% carrageenan. Biochemical and immunological analyses were performed at the end of the study. We observed no adverse effects of Jonlon-5at doses up to were observed at doses up to 2000 mg/kg/day. In the sub-acute study, there was a significant increase (p<0.05) in the activity of serum liver enzymes of rats administered with 500 mg/kg body weight of Jonlon-5. The results demonstrate that Jonlon-5 can significantly reduce paw edema and inhibit the production of MDA, tumor necrosis factor-α, and interleukin-1β (p<0.05). Our findings suggest that Jonlon-5 has anti-inflammatory effects in carrageenan-induced in rats with arthritis. However, intake of high doses may exhibit mild liver toxicity.

Keywords: Traditional medicine, Jonlon-5, Carrageenan, Acute inflammation, Acute toxicity

*Corresponding author

      The number of people with arthritis is rising rapidly around the world1-2. Arthritis is one of the leading causes of disability among adults and is very costly in both personal and economic terms2. Despite many advances in the management of arthritis, progress on finding a cure remains elusive. This is because currently available anti-arthritis medications have adverse effects with long-term use, creating the need for more effective and safe treatment options.  An alternative with some advantages, such as fewer side effects and lower cost, is herbal medicine3

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     In traditional Mongolian medicine, Jonlon-5, a mixed herbal medicine, is used to treat fever, infection, arthritis,  and heart disease4. Jonlon-5 tastes bitter and has a cool potency. It is composed of five herbs: Radix Sophoroealopecuroides, Herba Gentianaebarbatae, Fructus Gardeniaejasminoides, Fructus Terminaliaechebulae, and Fructus Terminaliaebelliricae. Previous phytochemical studies have revealed that Jonlon-5 contains alkaloids (mainly matrine and oxymatrine), flavonoids, saponin, and organic oxides5.

      S. alopecuroides L (Unegen suulkhei lider), the main compound in Jonlon-5, is rich in alkaloids and flavonoids and has anti-inflammatory, anti-oxidative, and anti-bacterial effects6-7. G. barbatae (Sormuust degd) and its main active alkaloids, flavonoids, xanthone, and secoridoids have been found to have anti-inflammatory, hepatoprotective, and anti-oxidative effects7-10. G. jasminoides (Arur) containing geniposide, genipin, and crocetin has a broad spectrum of therapeutic effects that include anti-oxidative, anti-inflammatory, anti-thrombosis, and anti-atherosclerosis activity11. Evidence from previous pharmacological and ethnomedical studies have shown that T. chebula (Jurur) exhibits anti-bacterial, anti-inflammatory, immunomodulatory, hypoglycemic, anti-oxidative, and analgesic effects, and confers hepatic protection12-13. T. bellirica (Barur) has been reported to have analgesic, anti-inflammatory, anti-secretory, and hepatoprotective activities14-15. The latter two plants are rich in gallic acid, which has strong anti-oxidative activity in human tissues16.

     The traditional approach of combining several herbal medicines has been found to be more effective than administering them in isolation17. Given evidence that Jonlon-5 and its herbal components have potential antibacterial, anti-inflammatory, and anti-oxidative activities, we hypothesized that Jonlon-5 could be a safe and effective medication against inflammation. In the present study, we investigated the anti-inflammatory activity of Jonlon-5 on rats that were induced with paw edema. The acute and sub-acute toxicity of Jonlon-5 was also evaluated.

Material and Methods

Preparation of Jonlon-5

     Jonlon-5 herbal medicine was prepared in the Institute of Traditional Medicine and Technology of Mongolia (Table 1). The crude components of Jonlon-5 were authenticated by experts from Department of Botany, Institute of Biology, Mongolia. One gram of crushed dried plant material was dissolved into 100 ml of distilled water and boiled at 100°C for 15 min. The prepared solution was used for the tests.

Experiment animals

     The current study was performed on Wistar albino rats of both sexes weighting 200 to 250 g at the pharmacology laboratory of the Institute of Traditional Medicine and Technology of Mongolia. The animals were kept and followed at standard laboratory conditions (room temperature (20 ± 2°C) and 12 h/12 h light/dark cycle). They were fed a commercial pellet diet and sterilized tap water.

     For the acute study, healthy white mice of both sexes weighting­­­ 22–28 grams were purchased from the animal house of the Institute of Traditional Medicine and Technology of Mongolia and used after one week of quarantine. All experiments were carried out in accordance with the institutional guideline for the Care and Use of Laboratory Animals. This study was approved by the Ethics Committee of the Mongolian National University of Medical Sciences.

Acute toxicity study

     In animals, a single oral administration of Jonlon-5 did not produce any toxicity at a dose of up to 2000 mg/kg. Accordingly, a dose of 2000 mg/kg was used as the maximum dose as recommended by the Organization for Economic Co-operation and Development (OECD) test guidelines. Thirty mice of each sex were randomly assigned to two groups with fifteen mice of each. The animals in treatment group received a single dose of 2000mg/kg via gavage. Control mice were given an equal amount of distilled water. After oral administration, all clinical signs were recorded before and after dosing at 1, 2, 4, and 24h.

Sub-acute toxicity study

     For the sub-acute toxicity study, 40 albino rats were randomly grouped into four groups of ten rats each. The control group was administered with 0.5 ml of saline once daily for 28 days. The rats in the other three groups were orally treated with 100, 200, and 500 mg/kg body weight of Jonlon-5, respectively, for 28 days. The rats were under constant daily observation for any signs of toxicity, and their body weights were also recorded weekly throughout the experimental period. On the 29th day of the experiment, all the animals in the four groups were anesthetized under ketamine hydrochloride (80 mg/kg). Blood samples were collected by cardiac puncture into non-heparinised bottles for biochemical investigation and centrifuged according to groups. The serum was separated into sterile bottles for biochemical analysis.

Carrageenan-induced inflammation

     The animals were randomized into four groups (ten in each group): normal control (NC), inflammation, indomethacin (IMC), and Jonlon-5 (Jonlon-5 at 100 mg/kg of body weight) groups. The rats in the control and inflammation groups were administered distilled water orally (5 mL/kg of body weight). The rats in the IMC group were given oral indomethacin (10 mg/kg of body weight) 2 h prior to the induction of arthritis. Indomethacin has been widely used for arthritis treatment of because of its anti-inflammation and analgesic activities. The Jonlon-5 group received Jonlon-5 (100 mg/kg of body weight) 2 h prior to the induction of inflammation. Inflammation was induced by 0.1 ml 1% carrageenan. Paw volume was measured at baseline, 30, 60, 120, and 240 min after induction with a plethysmometer. After 4 h, the rats were sacrificed, and blood was collected for various analyses.

Biochemical analysis

     Determination of the level of malondialdehyde (MDA) was determined using the fluorimetric method according to a thiobarbituric acid test. In brief, 0.5 ml of plasma was heated in a boiling water bath for 15 min with a solution of 0.68% 2.5 ml thiobarbituric acid and incubated for 15 min on ice. The mixture was centrifuged at 12000 rpm for 5 min at 4°C. The MDA was spectrofluorometrically determined using a synchronous technique with excitation at 532 nm.

Plasma levels of cytokines

     After being allowed to stand for 30 min, the blood was centrifuged at 3000 rpm/min for 10 min at 4°C; then, plasmas were collected for analysis. The serum concentrations of the tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) were determined using a commercial enzyme-linked immunosorbent assay (ELISA) method (ELISA kit, Republic of China).

Statistical Analyses

     The data are expressed as the mean ± standard deviation. A one-way ANOVA was used to identify the differences between groups, and followed with Tukey’s test. Statistical significance was set at the level of  p<0.05. The statistical analyses were carried out using the Statistical Package for the Social Sciences (SPSS) version 20.0 for Windows.


Acute toxicity of Jonlon-5

     We evaluated the acute toxicity of Jonlon-5. There were no significant differences in body weight change findings between the Jonlon-5 and NC groups, and no clinical signs were observed in the Jonlon-5 group.

Sub-acute toxicity

     At the end of the experiment, there was a slight weight reduction in the Jonlon-5 treated rat groups compared to the control, but it was not statistically significant. This indicates the healthy status of the rats following Jonlon-5 decoction. Serum aminotransferases (ALT and AST) and alkaline phosphatase (ALP) activity significantly increased in a dose-dependent manner when the rats received various doses of Jonlon-5. ALT and AST activities were significantly higher in the 500 mg/kg body weight treated group when compared with the others (p<0.05). ALP activity showed no significant difference compared with the others (Fig. 1).

Effects of Jonlon-5 on carrageenan-induced arthritis

     The injection of carrageenan in the three groups led to a time-related increase in size of paw, which reached a maximum at 4 h. Compared to the control group, pretreatment of Jonlon-5 at a concentration of 100 mg/kg of body weight significantly (p< 0.05) reduced the development of paw edema, as shown in Fig. 2.

Effects of Jonlon-5 on MDA concentration

     The level of MDA which is a product of lipid peroxidation, was significantly elevated in the inflammation group as compared those in the NC group. However, the IMC group exhibited a significant fall in MDA levels compared with the inflammation group. In addition, the Jonlon-5 group showed a significant decrease in the level of MDA (p<0.05) compared to other groups (Fig. 3).

Effects of Jonlon-5 on the expression of pro-inflammatory cytokines of plasma

    The in vivo anti-inflammatory activity of Jonlon-5 was also monitored by evaluating the levels of TNF-α and IL-1βby ELISA. As shown in Figure 4, the TNF-α and IL-1β levels increased significantly in plasma after carrageenan-induced inflammation compared with the NC group. However, pretreatment with Jonlon-5 (100 mg/kg of bodyweight) decreased the production of TNF-α and IL-1β in plasma and was significantly different compared to other groups (p<0.05). Interestingly, Jonlon-5 at 100 mg/kg of body weight significantly suppressed the production of TNF-α and IL-1β, which was comparable to the effect of the non-steroid anti-inflammatory medication Indomethacin (10 mg/kg of body weight).

Discussion and Conclusion

     In traditional medicine, combinations of multiple herbs are used to increase their pharmacological activity and reduce adverse reactions17. Thus, in this study, we focused on whether the combination of herbs in Jonlon-5 has anti-inflammatory and anti-oxidative activities that are associated with a reduction in oxidative stress and inflammation and whether it can be used safely.

     The acute toxicity study allowed us to determine the safety of the materials. The study indicated that Jonlon-5 is a safe agent when given orally to rats in a single dose at a level of 2000 mg/kg. Moreover, no clinical signs of adverse events were observed. According to the Guidance on Acute Oral Toxicity Testing based on an oral LD50 value that was recommended by the OECD, Jonlon-5 may be assigned as class 5 (LD50>2000 mg/kg body weight), which was appeared to be the low toxicity class18.

     Administration of Jonlon-5 at two different doses (100 mg/kg and 200 kg/mg body weight) for 28 days had no statistically significant effect on serum levels of ALT and AST. The current study showed that Jonlon-5 up to 200 mg/kg body weight did not indicate any toxicity in the liver for the parameters used, ALT and AST. However, serum ALT and AST levels were found to increase significantly in rats receiving 500 mg/kg body weight of Jonlon-5 when compared to the control group. The significant increase in ALT and AST suggests that administration of high doses of Jonlon-5 may induce destruction of liver cells.

     Carrageenan is often used to produce non-immune-mediated inflammation when evaluating components with potential anti-inflammatory activity. In the current study, anti-inflammatory activity of Jonlon-5 is observed 30 min after carrageenan induction compared to the inflammation group. Our results demonstrate that Jonlon-5 has an anti-inflammatory effect via reduction of paw edema that is associated with carrageenan induction. In line with our results, previous studies have shown that the administration of Sendeng-4 (Fructus Terminaliaechebulae, Fructus Terminaliaebelliricae, Fructus Gardenia jasminoides, and Fructus Toosendan), and Lider-7 (Radix Sophoroealopecuroides, Radix Inulaehelenium, Fructus Gardeniae, Fructus Terminaliaebelliricae, Fructus T. chebulae, Herba Gentianaebarbatae, and Herba Lagotisintegrifoliae), which have similar components as Jonlon-5, exhibited an anti-inflammatory effect on both acute and chronic experimental inflammation19-21.

     According to the literature, carrageenan induced inflammation is associated with free radicals that lead to elevated lipid peroxidation6. MDA, a product of lipid peroxidation, which can be used as a biomarker of oxidative stress in tissues and plasmas. In the current study, we found that Jonlon-5 attenuated the rise in MDA level in plasma, indicating that Jonlon-5 can weaken the process of lipid peroxidation implicated in the pathogenesis of carrageenan induced inflammation. 

     TNF-α and IL-1β are inflammatory cytokines that are produced during inflammation that play an important role in host defenses. Previous studies have demonstrated an inhibition effect of different traditional medical treatments on the levels of TNF-α and IL-1β. Wen et al. reported that traditional Chinese herbs inhibited TNF-α, IL-1β, and IL-6 as well as levels of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in a carrageenan-induced paw edema22. Similarly, our study showed that pretreatment with Jonlon-5 reduced levels of TNF- and IL-1βcytokines in plasma.

     However, our study has some limitations. There was no histological examination to confirm the therapeutic effect of Jonlon-5 in an experimental model of arthritis. Another limitation of the study is the inability to include various doses and hence to determine the dose-dependent anti-inflammatory effect. This would have made the study more comprehensive, although the safe dose the authors chose provided a positive result.

     In conclusion, our findings suggest that Jonlon-5 is a safe drug with a potent in vivo anti-inflammatory effect in an acute inflammation animal model. We revealed that Jonlon-5 can suppress the production of MDA, TNF-α, and IL-6 in plasma. Jonlon-5 is a promising anti-inflammatory agent against inflammation but can cause mild liver damage at high doses.


     The authors would like to thank all staffs of the pharmacology laboratory and animal house, the Institute of Traditional Medicine and Technology, Mongolia, for their valuable contribution to the successful completion of the study and Mr Turner for language editing.


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Table 1 — Crude components of Jonlon-5 (1 g)

Mongolian name

Scientific name

Used part

Amount (g)

Unegen suulkhei lider

Sophora alopecuroides L.




Terminalia chebula




Gardenia jasminoides




Terminalia bellirica



Sormuust degd

Gentiana barbata



Total amount


Fig. 1 — Effect of Jonlon-5 on the activity of serum liver enzymes

Normal control group: n=10; Jonlon-5 100 mg/kg body weight group: n=10; Jonlon-5 200 mg/kg body weight group: n=10; Jonlon-5 500 mg/kg body weight group n=10. Data are reported as mean ± SD. Data with * are significantly different (p<0.05)

Fig. 2 — Effect of Jonlon-5 on Carrageenan-induced rat paw edema

Normal control group: n=10; Inflammation group n=10; Indomethacin 10 mg/kg body weight group: n=10; Jonlon-5 100 mg/kg body weight group: n=10; Data with * are significantly different (p<0.05)

Fig. 3 — Effect of Jonlon-5 on the level of malondialdehyde (MDA)

Control (inflammation) group: n=10; Indomethacin 10mg/kg body weight group: n=10; Jonlon-5 100 mg/kg body weight group: n=10. Data are reported as mean ± SD. Data with * are significantly different (p<0.05).

Fig. 4 — Effect of Jonlon-5 on the expression of tumor necrosis factor (TNF-α) and interleukin (IL-1β) protein in plasma. Normal control group: n=10; Indomethacin 10 mg/kg body weight group: n=10; Jonlon-5 100 mg/kg body weight group: n=10. Data are reported as mean ± SD. Data with * are significantly different (p<0.05)


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