1.       Introduction

Traditional Mongolian Medicine (TMM) has developed over thousands of years and greatly influenced by nomadic lifestyle and frequent exposure to severe climate conditions [1-3]. In TMM, decoction is most widely used form and preferable way of preparing decoction is boiling of the drugs with water. This helps in selective release of water-soluble active components making it less toxic. Moreover, such aqueous extracts are effective, absorbs quickly and completely with high bioavailability. However, the decoction is trouble to prepare, and inconvenient to carry and store [4]. Because of these different forms such as granules, capsules, tablets and syrups are developed as alternatives to decoctions and used widely by traditional practitioners in many parts of the world including China, Japan, Korea, US and some European countries.

In traditional medicine, acute lung injury is considered as hot-natured condition caused by impure blood, bile and microbes [5,6]. Deva-5 decoction is one of the multi-component compounds used for treatment of hot-natured conditions in traditional Mongolian medicine [5]. It is composed from 5 ingredients including Gentiana decumbens L., Momordica cochinchinensis L., Chiazospermum erectum Bernh., Polygonum bistorta L. and Terminalia chebula Retz [5,6]. Main components of Deva-5 have been shown to have antiviral, antibacterial and anti-inflammatory properties [7-15]. From our recent study on anti-viral effect of Deva-5, we found that Ch.erectum, T.chebula and M.cochinchinensis have high anti-viral activity against H3N8 and might be a promising potential source of new anti-viral agents [16].

De-Mon is syrup medicine produced with the ingredients of traditional recipe named Deva-5. Based on our previous study results, we postulated that De-Mon syrup could protect against LPS-mediated Acute Lung Injury (ALI). In the present study, we tested this hypothesis using a rat model of LPS-mediated ALI.

2.       Material and methods

2.1    Preparation of De-Mon

The crude herbal medicines from Gentiana decumbens L., Momordica cochinchinensis L., Chiazospermum erectum Bernh., Polygonum bistorta L. and Terminalia chebula Retz. were purchased from Traditional Drug Factory at the Institute of Traditional Medicine and Technology of Mongolia. All plant materials were size reduced into coarse powder (3 mm) and macerated with 20% ethanol. Then the extract was filtered out and concentrated under vacuum using rotary vacuum evaporator. The residue obtained was then subjected for further extraction processes. Herbal syrup was prepared according to standard formulae and all evaluation techniques have been carried out as per standards.

2.2    Reagents

Escherichia coli 055:B₅ endotoxin from Sigma-Aldrich and the cytokine immunoassay kits from Shanghai MLBIO Biotechnology Co. Ltd. (China) were used in the study. All other solvents and chemicals were of analytical grade.               

2.3    Experimental animals

A total of fifty 8-10-week-old male Wistar rats (180-220 g) were used in this study. All experimental animals were obtained from the Experiment Animal House, Institute of Traditional Medicine and Technology. The rats were housed in cages and maintained at room temperature with a 12-h light/dark cycle. They were fed with standard pellet diet and tap water ad libitum.

2.4    Experimental protocols

Rats were randomized into three groups: control group (n=20), LPS group (n=20), in which LPS (7.5 mg/kg dissolved in 0.5 mL sterile saline) was administered by an intravenous injection (iv) via the tail vein; and LPS+ De-Mon syrup group (n=20), in which De-Mon syrup (39.6 mg/kg, orally) was given for 5 days before injection of LPS (7.5 mg/kg dissolved in 0.5 mL sterile saline, iv) orally [17]. Rats were euthanized with an overdose of ketamine hydrochloride (80 mg/kg, ip). Lung tissue specimens and blood samples were then obtained for further analysis.

2.5    Plasma levels of cytokines (TNF-a and IL-1β, IL-6, IL-10)

Blood samples were collected via cardiac puncture at 3, 6, 9 and 12 h after the administration of LPS and from healthy rats. All rats were euthanized with ketamine hydrochloride before blood collection. The collected blood samples were centrifuged at 377.3 g for 10 min at 4^°C, and the plasma supernatant was stored at -20^°C until further analysis. The plasma levels of TNF-a and IL-1β, IL-6, IL-10, were detected using solid-phase sandwich enzyme-linked immune sorbent assay (ELISA, Shanghai MLBIO Biotechnology Co. Ltd.) kits specific for the detection of these factors, and the absorbance was measured at 450 nm by a plate reader (Chromate 4300 microplate, Shanghai MLBIO Biotechnology Co. Ltd., China).

2.6    Histological analysis

Twenty-four hours after LPS administration, the rats were euthanized (n=5, 3, and 5 in the control, LPS, and LPS +De-Mon groups, respectively). The obtained lung tissue specimens were fixed with 10% formalin, embedded in paraffin, cut into 5-mm thick sections and mounted onto slides. The sections were then stained with hematoxylin and eosin (H&E) according to the standard staining method [18]. Histologic changes were graded by a pathologist blind to the clinical status of the rats. Then the lung tissue samples were scored for the degree of intra-alveolar edema, intra-alveolar hemorrhage, and neutrophil infiltration using grades 0 to 4 (0, absent; 1, mild; 2, moderate; 3, severe; 4, overwhelming) with a maximum score of 12, as described previously [19].

2.7    Statistical analysis

Data are reported as mean±SD. Statistical significance was determined by one-way analysis of variance followed by Tukey’s multiple comparison test. A P value <0.05 was considered statistically significant.

3.       Results

3.1    Effects of De-Mon on serum level of TNF-α, IL-1β, IL-6 and IL-10

The in vivo anti-inflammatory activity of De-Mon was monitored by evaluating the levels of inflammatory cytokines. The levels of TNF-α, IL-1β and IL-6 level increased significantly in plasma after LPS administration compared with the control group and peaked at 3 h and 9 h respectively. Pre-treatment with De-Mon significantly suppressed the production of TNF-α (LPS + De-Mon group vs LPS group: p<0.01 at 3 h and 9 h), IL-6 (LPS + De-Mon group vs LPS group: p<0.05 at 9 h) and IL-1β (LPS + De-Mon group vs LPS group: p<0.01 at 3 h, 6 h, 9 h and 12 h) at the indicated time points (Figures 1A-C). Meanwhile, treatment with De-Mon induced the LPS-dependent IL-10 anti-inflammatory cytokine. The levels of IL-10 increased gradually and reached a peak at 12 h and was significantly different compared with the LPS group (LPS + De-Mon group vs LPS group: p<0.01 at 3 h, 6 h, 9 h and 12 h) (Figure 1D).

3.2    Pre-treatment with De-Mon attenuated LPS-mediated lung histopathological changes

To evaluate the histological changes, lung tissues harvested at 24 h after LPS administration, were stained with H&E. As shown in Figure 2A,B, lung tissues from the control group showed normal structure and no histological alterations. In the LPS-group, the lung tissues had significant histo pathological changes, such as thikening of the alveolar wall, bleeding and inflammatory cell infiltration (Figures 2C,D). Rats pre-treated with De-Mon showed less inflammation and distortion of lung architecture indicating that acute lung injury was attenuated by treatment (Figures 2E, F).

A, B, Histological feature of lung in health group. C, D, Histological feature of lung in control (LPS) group. E, F, Histological feature of lung in experimental (LPS+De-Mon) group.1, Alveolar wall.2, Alveoli.3, Bronchiole. Staining: Hematoxylin and eosin, x40, x100.

4.       Discussion

In traditional Mongolian medicine, acute lung injury is considered as hot-natured condition caused by impure blood, bile and microbes [5]. Deva-5 is one of the multi-component compounds used for treatment of hot-natured conditions in traditional medicine [5-6]. In current study, the protective effect of De-Mon, syrup version of traditional medicine Deva-5 on LPS-induced Acute Lung Injury (ALI) or Acute Respiratory Distress Syndrome (ARDS) was evaluated. Our data demonstrated that pre-treatment with De-Mon decreased pro-inflammatory cytokines production and attenuated lung tissues damage. This suggests that De-Mon might be potential therapeutic candidate for the ALI.

LPS-mediated ALI is characterized by pro-inflammatory cytokines activation which play important role in development and progression of ALI. IL-1β, for example, inhibits fluid transportation across the distal lung epithelium to cause surfactant abnormalities and increases protein permeability across the alveolar-capillary membrane. The levels of TNF-α, IL-1β and IL-6 increased in patients with ALI or ARDS [20,21]. These cytokines initiate and amplify the inflammatory response in ALI. In this study, we found that De-Mon significantly attenuated the production of TNF-α, IL-1β, and IL-6 and induced IL-10 production in LPS-mediated Acute Lung Injury. Therefore, our results suggested that the protective effects of De-Mon on LPS-mediated Acute Lung Injury may be attributable to the inhibition of inflammatory cytokines and induction of IL-10.

Anti-inflammatory properties of De-Mon components have been studied at some degree by others. Triterpenoidal saponin isolated from the seeds of Momordica cochinchinensis inhibits LPS-induced expression of IL-6 in macrophages. Quillaic acid glycoside isolated from the seeds of Momordica cochinchinensis inhibit LPS-induced expression of NO and IL-6 in macrophages nuclear factor-κB pathway. Protopine isolated from Chiazospermum erectum attenuates LPS-induced inflammatory responses in murine macrophages, and reduced the production of nitric oxide, inducible nitric oxide synthase, cyclooxygenase-2, and pro-inflammatory cytokines such as TNF-α, IL-1 and IL-6 via the inhibition of phosphorylation of extracellular-signal-regulated kinases 1/2 and c-Jun N-terminal kinase and inhibition of nuclear factor-κB activation in LPS-activated macrophages [22]. From our previous study on anti-viral effect of Deva-5, we found that Ch.erectum, T.chebula and M.cochinchinensis which are main components of De-Mon have high anti-viral activity against H3N8 and might be a promising potential source of new anti-viral agents [16]. Ch.erectum, T.chebula and M.cochinchinensis are rich in alkaloids and flavonoids, thus, we speculate that these components had a major contribution to the effects observed in this study.

In conclusion, De-Mon syrup, developed from the traditional Mongolian recipe reduces lung inflammation by inhibiting TNF-α, IL-1β and IL-6 and increasing activity of IL-10 in rats administered LPS. Our results suggest that De-Mon could be a potential medicine for preventing and treating LPS induced ALI.

5.       Competing interests

The authors declare that they have no competing financial interests.

6.       Acknowledgements

This study was supported by the Mongolian Foundation for Science end Technology (MFST; agreement: IN_A/178-10/2016).

Figure 1: Effects of De-Mon on the production of plasma levels of TNF-α, IL-6, IL-1β and IL-10 after LPS administration. Rats were given De-Mon orally during 5 days prior to an i.v. administration of LPS. Blood was collected at 3 h, 6 h, 9 h and 12 h following LPS challenge to analyze the cytokines of TNF-α (A), IL-6 (B), IL-1β (C) and IL-10 (D). The values presented are mean ± SD (n=20 in each group). ^#p<0.01, control (LPS) group compared to health group; * p<0.05 De-Mon + LPS group vs. LPS group.

Figure 2: Histological feature of lung tissue after 24 hours since LPS administration.

1.       Pitschmann A, Purevsuren S, Obmann A, Natsagdorj D, Gunbilig D, et al. (2013) Traditional Mongolian Medicine: history and status quo. Phytochemistry reviews 12: 943-959.

2.       Shagdarsuren C: Die traditionelle Mongolische Medizin. Die Mongolen Pinguin-Verlag, Innsbruck Google Scholar 1989.

3.       Vargas-O'Bryan IM, Xun Z: Disease, religion and healing in Asia: Collaborations and collisions 14.

4.       Shang E, Zhu Z, Liu L, Tang Y, Duan J-a (2012) UPLC-QTOF-MS with chemical profiling approach for rapdly evaluating chemical consistency between traditional and dispensing granule decoctions of Tao-Hong-Si-Wu decoction. Chemistry Central Journal 6: 143.

5.       Tumurbaatar N: Hot-Natured Disorders in Traditional Mongolian Medicine. (1998) In.: Eruul enkh press, Ulaanbaatar 1998.

6.       Traditional Medical source book: Manag rinchin junai.

7.       Ligaa U, Davaasuren B, Ninjil N (2005) Mongolian medicinal plants using in Western and Eastern medicine. Ulaanbaatar. JKC printing 2005: 121-122.

8.       World Health Organization: Medicinal Plants in Mongolia. In., edn. Manila: WHO Regional Office for the Western Pacific 2013: 97-196.

9.       Su Y, Li S, Li N, Chen L, Zhang J, et al. (2011) Seven alkaloids and their antibacterial activity from Hypecoum erectum L. Journal of Medicinal Plants Research 5: 5428-5432.

10.    Bae D-S, Kim Y-H, Pan C-H, Nho C-W, Samdan J, et al. (2012) Protopine reduces the inflammatory activity of lipopolysaccharide-stimulated murine macrophages. BMB reports 45: 108-113.

11.    Badmaev V, Nowakowski (2000) Protection of epithelial cells against influenza A virus by a plant derived biological response modifier Ledretan‐96. Phytotherapy Research 14: 245-249.

12.    Bag A, Bhattacharyya SK, Pal BNK, Chattopadhyay RR (2009) Evaluation of antibacterial properties of Chebulic myrobalan (fruit of Terminalia chebula Retz.) extracts against methicillin resistant Staphylococcus aureus and trimethoprim-sulphamethoxazole resistant uropathogenic Escherichia coli. African Journal of Plant Science 3: 025-029.

13.    Myagmar B-E, Aniya Y (2000) Free radical scavenging action of medicinal herbs from Mongolia. Phytomedicine 7: 221-229.

14.    Jung K, Chin Y-W, Yoon Kd, Chae H-S, Kim CY, et al. (2013) Anti-inflammatory properties of a triterpenoidal glycoside from Momordica cochinchinensis in LPS-stimulated macrophages. Immunopharmacology and immunotoxicology 35: 8-14.

15.    Tsoi AYK, Ng TB, Fong WP (2005) Antioxidative effect of a chymotrypsin inhibitor from Momordica cochinchinensis (Cucurbitaceae) seeds in a primary rat hepatocyte culture. Journal of peptide science 11: 665-668.

16.    Oyuntsetseg N, Khasnatinov MA, Molor-Erdene P, Baigalmaa J, Chimedragchaa C, et.al. (2014) Evaluation of direct antiviral activity of the Deva-5 herb formulation and extracts of five Asian plants against influenza A virus H3N8. BMC Complement Altern Med 14: 235.

17.    G. Li, C.L. Zhou, Q.S. Zhou, H.D. Zou (2016) Galant amine protects against lipopolysaccharide induced acute lung injury in rats. Brazilian Journal of Medical and Biological Research 49.

18.    Imanaka H, Shimaoka M, Matsuura N, Nishimura M, Ohta N, et al. (2001) Ventilator-induced lung injury is associated with neutrophil infiltration, macrophage activation, and TGF-beta 1 mRNA upregulation in rat lungs. Anesth Analg 92: 428-436.

19.    Chen F, Liu Z, Wu W, Rozo C, Bowdridge S, et al. (2012) An essential role for TH2-type responses in limiting acute tissue damage during experimental helminth infection. Nat Med 18: 260-266.

20.    Ajiboye O, Segal JB (2017) National trends in the treatment of diabetic nephropathy in the United States. J Clin Pharm Ther 42: 311-317.

21.    Giebelen IA, van Waterloo DJ, LaRosa GJ, de Vos AF, van der Poll T (2007) Local stimulation of α7 cholinergic receptors inhibits LPS-induced TNF-α release in the mouse lung. Shock 28: 700-703.

22.    Deok SB, Young HK, Cheol-HP, Chu WN, Javzan S, et al. (2012) Protopine reduces the inflammatory activity of lipopolysaccharide-stimulated murine macrophages. BMB reports 45: 108-113.