Activities of Antitussive of even Alkaloids from Bulbus Fritillariae cirrhosae

The objective of this study was to isolate active alkaloids from the Bulbus Fritillariae Cirrhosae (BFC), to evaluate their anti-tussive to find the highly active substance from the BFC for further new drug development. Materials and methods: The BFC was extracted with chloroform: methanol and alkaloids were isolated by phytochemical methods. The anti-tussive activity was investigated using ammonia induced mice coughing. Results: The seven alkaloids (1)∼(7) were isolated from the BFC and the structure were determined. The seven alkaloids significantly inhibited mice’s frequency of cough induced by ammonia, increased mice’s latent period of cough and show activity dose-dependently. The inhibitions of cough number of these alkaloids are 65.33% (1), 60.28% (2), 35.06% (3), 37.26% (4), 56.37% (5), 57.80% (6), 59.07% (7). The results of the present study provide a convincing proof to put BFC as one of the most popular antitussive Traditional Chinese Medicine (TCM) in China. *Corresponding author: Dongdong Wang, Department of Pharmacogonosy, West China College of Pharmacy, Sichuan University, No. 17, Duan 3, Renmin Nan Road, Chengdu 610041, P. R. China, Tel: +86-028-85503950; Fax: +86028-85503950; E-mail: wddong1988@hotmail.com Received May 29, 2014; Accepted August 18, 2014; Published August 20, 2014 Citation: Wang D, Wang S, Chen W (2014) Activities of Antitussive of even Alkaloids from Bulbus Fritillariae cirrhosae. Nat Prod Chem Res S1:005. doi:10.4172/23296836.S1-005 Copyright: © 2014 Wang D, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
Coughing is a symptom of respiratory illness which causes chest and thorax pain [1]. Presently available therapies to treat cough are limited for lack of effective and safe medications. Moreover, most existed antitussive drugs could bring inevitable or intolerable side effects [2]. In TCM, many plants are recorded to treat respiratory complaints such as cough, bronchial inflammation, pneumonia and expectoration [3], which have been used for hundreds of years. So it is a noticeable path to search for effective medicines to treat cough in the rich traditional Chinese medicine in the world [4]. BFC, is a well known antitussive TCM used for a long time. It has been used in clinic for more than 2000 years in China due to the positive potent therapeutic effects, low toxicity and minimal side effects. The pharmacological studies indicate that the crude alkaloids extract of BFC have good antitussive activities [5,6]. Alkaloids were established to be the major biologically active components in the genus of Fritillaria. BFC which has been considered as the most valuable medicine in the genus of Fritillaria lacks further phytochemical study and pharmacological research of monomers of alkaloids in vivo. So we focused our studies on the phytochemical and pharmacological research on BFC. The isolation of alkaloids, evaluation of the antitussive activities of different alkaloids are the subject of this paper.

Extraction and isolation
Dried ground bulbs (15 kg) were powdered and treated in ammonia (1200 ml) for 15 hours, then extracted with chloroform to obtain crude extract (38 g). TAF was obtained by using previously described methods [7,8]. The concentrated extract was dissolved in 3% HCl and partitioned with chloroform. The pH of the aqueous solution was readjusted with NH 4 OH to 10.0 and extracted with CHCl 3 . The CHCl 3 extract (crude alkaloid, 9.5 g) and was chromatographed over Si gel using petroleum ether-acetone-diethylamine (6: 1: 2∼1: 1: 2) of increasing polarity as eluent to obtain seven fractions [9].

Animals and administration
Kunming mice (18-22 g) were purchased from Experimental Animal Center, West China College of Pharmacy, Sichuan University (Chengdu, China). All animals were housed at room temperature (22-24℃) and constant humidity (40-70%) under a 12 h light-dark cycle and provided with food and water ad libitum.

Anti-tussive activity
Kunming mice were randomly divided into 9 groups of 6 each and administered according to Section 2.3. Anti-tussive activity was investigated on a classical mouse cough model induced by ammonia liquor [10,11]. Briefly, each mouse was placed in a 1000 ml special glass chamber and exposed to, 0.3 ml 25% NH 4 OH for 45 seconds. Then the frequency of the cough for 5 min and latent period of cough were observed. The number of coughs produced 60min after the administration of antitussive agents (C t ) was compared with the number of control coughs (C 0 ). The antitussive effect was expressed as the percentage of inhibition of the number of control coughs [(C 0 −C t )/ C 0 ×100%]. of mean (S.E.M.). One-way analyses of variance (ANOVA) test was performed for multiple group comparison and Student's t-test was carried out for direct comparison of two groups. Values of p<0.05 imply significance of the pharmacological effects in the experiments.

Effect of alkaloids on the ammonia liquor induced cough in mice
The effects of the alkaloids on the ammonia liquor induced cough in mice are shown in Table 1. All alkaloids showed potent anti-tussive effects. Positive and tested groups could enhance latent period of cough and all the increase presented significantly, compared with the period of Control.

Discussion and Conclusion
The BFC is one of the most popular antitussive and expectorant TCM in Chinese communities. Potent antitussive, expectorant and antiasthmatic activities of the crude alkaloids extract from BFC were previously reported in the pharmacological studies [5,6,12]. In one word, the traditional knowledge and available clinical efficiency led us to isolate and characterize the active constituents present in BFC. In the present study, seven alkaloids were isolated from BFC.
In conclusion, the results of the present study provide a convincing proof to put BFC as one of the most popular antitussive and expectorant TCM in Chinese communities. Through the pharmacological evaluation on antitussive, expectorant and anti-inflammatory activities of all alkaloids respectively, (1), (2), (5), (6), (7) were highly active constituents of crude alkaloids extract from BFC. The mechanism of action explained for the observed activities have not been established, and thus further investigation needs conducted.