WO2009011499A1

WO2009011499A1 - Quantitative analysis method of pelargonium sidoides syrup or solution

Info

Publication number: WO2009011499A1
Application number: PCT/KR2008/003514
Authority: WO
Inventors: Jin Hang Kong; Yang Bae Kim; Chang Weon Yoon; Yong Jun Cah; Sang Seob Han; Chungkeun Rhee; Minho Oak
Original assignee: Dr. Willmar Schwabe Gmbh & Co.Kg
Priority date: 2007-07-18
Filing date: 2008-06-20
Publication date: 2009-01-22
Also published as: KR20090008728A

Abstract

The present invention relates to a quantitative analysis method for a syrup or solution preparation containing a Pelargonium sidoides extract which is used a therapeutic agent for acute or chronic infections, to perform a content analysis of the extract which is responsible for the manifestation of efficacy. The quantitative analysis method of the present invention has enabled product quality management and guaranteed manifestation of pharmacological effects through the content measurement of the Pelargonium sidoides extract in a syrup or solution, and the analysis method has an advantage of exhibiting excellent reproducibility and high reliability through relatively simple operations.

Description

QUANTITATIVE ANALYSIS METHOD OF PELARGONIUM SIDOIDES SYRUP OR SOLUTION

Technical Field

[1] The present invention relates to a quantitative analysis method for a syrup or solution containing a Pelargonium sidoides extract which is used as a therapeutic agent for an acute or chronic infection. Background Art

[2] An extract extracted from the roots of Pelargonium species such as Pelargonium sidoides and Pelargonium reniforme, which grow naturally in Southern Africa, has been widely used traditionally in Africa as a therapeutic agent for diarrhea, gastrointestinal complaints, dysmenorrhoea, liver diseases and the like (Watt, C. (1962) Medicinal and poisonous plants of southern and eastern Africa. Livingstone, Edinburgh, London, S. 449-455). In addition to that, the extract is known to have outstanding effects in the treatment of diseases in the respiratory system, and particularly tuberculosis.

[3] It is known that the Pelargonium sidoides extract which is known to be used originally in South Africa as a traditional medicinal material, has also been used as a medicinal material in other areas, specifically in the central part of Chile, Mexico and others. That is, in the above-described regions, the extract of Pelargonium sidoides has been used in remedy of diarrhea or various gastrointestinal syndromes, by making use of the astringent function possessed by the extract. These effects are known to be attributable to tannin, which is a constituent component of the extract (Jose San Martin, A. (1983) Econom. Bot., 37, 216-227; INI (1994) Atlas de las plantas de Ia medicina tradicional mexicans (Institute Naάonal Indigenista, Hrsg.) ^"VbI. II, Av. Revolution no 1279, Col. Tiacopac, Mexiko, S. 667 und 950).

[4] Moreover, in the early 20 century, a root extract of Pelargonium sidoides was used also in the United States, under the name of Umckaloabo, for the treatment of infection in the upper airway (Kauffer, HJ. (1915) Dental Cosmos 57, 1366), for example, rhinopharyngitis, tonsillitis, sinusitis, bronchitis and the like.

[5] These effects are known to be attributable to the antimicrobial effects of the

Pelargonium sidoides extract.

[6] M>re specifically, it was found through a coumarin test that the Pelargonium sidoides extract has a moderate degree of cytotoxicity against GLC4, lung cancer cells and COLO320 cells, and it was shown that such cytotoxic effect was totally dependent on the nature and position of the substituents on the aromatic rings. Further, the extract exhibited a moderate degree of bacteriostatic effects in an experiment performed through an agar dilution test using ooumarin and a tannin compound for the antimicrobial action against various Gram-positive and Gram-negative bacteria. The minimal inhibitory concentration (MIC) value is from 200 to 2000 μg/ml (in the case of Goumarin), and from 500 to 2000 μg/ml (in the case of monomer tannin precursor), and representative substances having a high degree of antibacterial action include ooumarin trioxide and tetraoxide. Such definite antibacterial action is exhibited by an extract, particularly an extract of the roots and leaves, of Pelargonium sidoides, and as for the solvent, water exhibits the greatest antibacterial effects (Kayser, O. (1997) Phenolic constituents of Pelargonium sidoides DC. and studies of the efficacy of the Umcka plant material (Pelargonium sidoides DC. and Pelargonium reniforme CURT.), Doctorial dissertation, Berlin Free University).

[7] Meanwhile, the Pelargonium sidoides extract is useful in the treatment of an acute or chronic infection, as the extract exerts influence on the immunoregulatory effects.

[8] Specifically, the Pelargonium sidoides extract is reported to enhance the synthesis of

TNF-α, INF-β and nitric oxide (NO) p. Kolodziej et al., Phytomediάne 10 (Suppl. 4), 18-24 (2003)).

[9] Moreover, investigations reported that coumarins derived from the Pelargonium sidoides extract exhibit immunostimulatory effects in various experimental models. Specifically, in an intracellular Leishmania infection model, these substances exhibited a moderate degree of effect in a structure-dependent manner in relation to the oxygen- dependent immune defense mechanism, and in particular, a moderate degree of stimulation of the defense mechanism to the intracellular infection via the NO production mediated by simple coumarin was shown to increase according to the degree of increase in oxygen generation. Furthermore, gallic acid, which is another constituent component of the extract, is an excellent derivative, whose activity is attributed to nitric oxide radicals (Kayser, O. (1997) Phenolic constituents of Pelargonium sidoides DC. and studies of the efficacy of the Umcka plant material ( Pelargonium sidoides DC. and Pelargonium reniforme CURT.), Doctorial dissertation, Berlin Free University).

[10] Such Pelargonium sidoides extract has been currently confirmed to be effective in acute or chronic infections, and particularly in infections in the respiratory system or otorhinolaryngeal areas, such as bronchitis, sinusitis, tonsillitis and rhinopharyngitis. [11] Meanwhile, syrup preparations or solution preparations are advantageous in that they can be easily administered to children or seniors who lack the ability to swallow tablets, they exhibit the effects of drug quickly compared to tablets, and they can reduce the differences in bioavailability which may be caused by the differences in drug solubility of individual preparations when administered in the form of tablets. Particularly in the case of syrup preparations, the bitter taste and unpleasant odor caused by the drug during oral administration can be dramatically improved, so that patients' compliance to the drug is enhanced, and the drug can be easily administered to children or seniors.

[12] Fbwever, in the case of syrups or solutions containing a Pelargonium sidoides extract, the content analysis method for the extract which is responsible for the manifestation of efficacy has not been established, and thus actual application thereof has been hindered.

Disclosure of Invention Technical Problem

[13] The present invention has been designed to solve the problems as described above, and it is an object of the invention to provide a quantitative analysis method for a syrup or solution containing a Pelargonium sidoides extract, to show the content of the extract which is responsible for the manifestation of efficacy. Technical Solution

[14] The quantitative analysis method for Pelargonium sidoides extract of the present invention is characterized, in order to achieve the object as described above, by comprising the following steps:

[15] (A) preparing a test solution by mixing 1 part by weight of a Pelargonium sidoides extract with 4) to 60 parts by weight of a 15 to 30 wt% aqueous ethanol solution, 6 to 9 parts by weight of Folin Ciocalteus phenol reagent, 4) to 60 parts by weight of a 10 to 20 wt% aqueous solution of sodium carbonate, and 15 to 4) parts by weight of water, centrifuging the mixture solution, and taking the supernatant as the test solution;

[16] (B) preparing a standard solution by mixing 1 part by weight of an aqueous ethanol solution of epicatechin with 8 to 12 parts by weight of a 15 to 30 wt% aqueous ethanol solution, 1 to 2 parts by weight of Folin Ciocalteus phenol reagent, 8 to 12 parts by weight of a 10 to 20 wt% aqueous solution of sodium carbonate, and 3 to 8 parts by weight of water, centrifuging the mixture solution, and taking the supernatant as the standard solution; [17] (C) measuring the absorbance of the test solution and the standard solution, while using water as a control; and [18] (D) calculating the total phenol content in terms of epicatechin by the following math figure 1: [19] MathFigure 1

[Math.l]

E _s ^χ V x 200 ^{X 1 O}°

[20] wherein Et is the absorbance of the test solution,

[21] Es is the absorbance of the standard solution,

[22] V is the volume of the test solution measured in m-6, and

[23] S is the mass of the standard solution measured in mg.

[24] A step of leaving the mixture solution to stand at normal temperature for 10 to 30 minutes before centrifuging the mixture solution, may also be additionally included. [25] The centrifuging is preferably performed for 5 to 20 minutes.

[26] The absorbance is preferably measured at 720 nm.

[27] The 15 to 30 wt% aqueous ethanol solution is preferably prepared by diluting a 95 to

96 wt% aqueous ethanol solution with water at a volume ratio of 1:2 to 5.

Advantageous Effects

[28] The quantitative analysis method for a sryup or solution containing a Pelargonium sidoides extract of the present invention has enabled product quality management and guaranteed manifestation of pharmacological effects through the content measurement of the Pelargonium sidoides extract in the syrup or solution, and the analysis method has an advantage of exhibiting excellent reproducibility and high reliability through relatively simple operations. Brief Description of the Drawings

[29] Fig. 1 is a graph showing the relationship between the concentration of a

Pelargonium sidoides extract and the absorbance. Best Mode for Carrying Out the Invention

[30] Hereinafter, preferred embodiments of the present invention will be described in detail. In the following description, a number of specific matters such as specific constituent elements are disclosed, but it will be obvious to those having ordinary skill in the related art that these matters are provided only for the purpose of helping in more general understanding of the present invention, and the present invention can be carried out without these specific matters. Further, in describing the present invention, when it is judged that specific descriptions on related known functions or constitutions may unnecessarily make the gist of the invention ambiguous, the detailed description will be omitted.

[31] The quantitative analysis method for Pelargonium sidoides extract of the present invention first starts with a step of preparing a test solution containing a Pelargonium sidoides extract, and a standard solution as the base to be compared with the test solution.

[32] First of all, the Pelargonium sidoides extract to be analyzed may be any material containing a Pelargonium sidoides extract, regardless of the dosage form, and according to the present invention, a syrup or solution preparation containing the extract is more preferred.

[33] 1 part by weight of said extract is mixed with 4) to 60 parts by weight of a 15 to 30 wt% aqueous ethanol solution, 6 to 9 parts by weight of Folin Ciocalteus phenol reagent, 4) to 60 parts by weight of a 10 to 20 wt% aqueous solution of sodium carbonate, and 15 to 4) parts by weight of water.

[34] Here, the 15 to 30 wt% aqueous ethanol solution is preferably prepared by diluting a

95 to 96 wt% aqueous ethanol solution with water at a volume ratio of 1:2 to 5.

[35] The mixture solution is subsequently centrifuged, and then the supernatant is taken as the test solution.

[36] At this time, the centrifuging is preferably performed for 5 to 20 minutes.

[37] In particular, in the present invention, it is more preferable that a step of leaving the mixture solution to stand at normal temperature for 10 to 30 minutes before centrifuging the solution, be additionally included from the viewpoint of enhancing the reproducibility of the analysis results.

[38] Apart from the test solution, an epicatechin standard solution is prepared. First, 1 part by weight of an aqueous ethanol solution of epicatechin prepared by appropriately diluting epicatechin with the above-described 15 to 30 wt% aqueous ethanol solution, is mixed with 8 to 12 parts by weight of the 15 to 30 wt% aqueous ethanol solution, 1 to 2 parts by weight of Folin Ciocalteus phenol reagent, 8 to 12 parts by weight of a 10 to 20 wt% aqueous solution of sodium carbonate, and 3 to 8 parts by weight of water.

[39] Hereinafter, the step of leaving to stand and the step of centrifuging are identical to those in the step of preparing a test solution.

[4)] Subsequently, the test solution and standard solution prepared as described above are subjected to measurement of the absorbance, using water as a control. The measurement is most preferably conducted at a wavelength of 720 nm. [41] Finally, the content of Pelargonium sidoides extract is determined by calculating the total phenol content in terms of epicatechin, by the following math figure 1. [42] [Math Figure 1]

[43, E _t * S

E _s ^χ V x 200

[44] wherein Et is the absorbance of the test solution,

[45] Es is the absorbance of the standard solution,

[46] V is the volume of the test solution measured in m#, and

[47] S is the mass of the standard solution measured in mg.

[48] Hereinafter, Examples of the present invention will be described.

Mode for the Invention

[49] Example 1 : Preparation of test solution

[50] 200 id of a syrup containing a Pelargonium sidoides extract, 10 m# of an ethanol dilution prepared by mixing and diluting a 96 wt% of aqueous ethanol solution with distilled water at 1:3, 1.5 m-6 of Folin Ciocalteus phenol reagent, and 10 m# of an aqueous solution of sodium carbonate were precisely weighed in a flask having a capacity of 25 m#, and distilled water was added thereto to a total volume of 25 mL This mixture solution was left to stand at normal temperature for 20 minutes, and then was centrifuged for 10 minutes. The supernatant was taken as the test solution.

[51] Example 2: Preparation of standard solution

[52] 10 mg of epicatechin was precisely weighed and introduced into a flask having a capacity of 20 m#, and an ethanol dilution prepared by mixing and diluting a 96 wt% aqueous solution of ethanol with distilled water at 1:3 was added thereto to a total volume of 20 mL 1 m# of the solution was taken, and the above-mentioned ethanol dilution was added again thereto to a total volume of 10 m-6. Then, another 1 mi was taken therefrom. 1 mi of the finally taken solution, 10 m# of the ethanol dilution, 1.5 il of Folin Ciocalteus phenol reagent, and 10 m# of an aqueous solution of sodium carbonate were precisely weighed in a flask having a capacity of 25 m#, and distilled water was added thereto to a total volume of 25 mL This mixture solution was left to stand at normal temperature for 20 minutes, and then centrifuged for 10 minutes. The supernatant was taken as the standard solution.

[53] Example 3: Measurement of absorbance (I)

[54] The test solution from Example 1 and the standard solution from Example 2 were subjected to the measurement of absorbance (Agilent, Inc., IVbdel 8453) at 720 nm, with a total path length of 10 mm, using water as a control. The measurements were repeated 5 times, and the results are presented in Table 1.

[55] Table 1 [Table 1] [Table ]

[56] The results of the measurements showed a high degree of reproducibility with a relative standard deviation of 1.14, as shown in the above Table 1.

[57] Example 4: Measurement of absorbance (2) [58] The process of Example 3 was repeated, except that meausrements were made while varying the amount of the syrup containing a Pelargonium sidoides extract of Example 1 to 50, 80, 100, 150 or 200 μt The results are presented in Table 2 and Fig. 1.

[59] Table 2 [Table 2] [Table ]

[60] As a result of the measurements, high linearity with a correlation coefficient of 0.99973 was confirmed. Thus, it can be utilized that at least within the above- mentioned concentration range, a proportional relationship exists between the con- centration and the absorbance.

[61] As discussed above, preferred embodiments of the present invention have been illustrated and explained, but the present invention is not intended to be limited to the above-described specific embodiments, and any person having ordinary skill in the pertinent art can definitely carry out various modifications without departing from the gist of the present invention. Therefore, the scope of the present invention should not be interpreted to be limited to the above Examples, and should be determined by the claims that will be described below as well as equivalents to these claims.

[62]

Claims

Claims[1] A quantitative analysis method for Pelargonium sidoides extract, comprising the following steps:(A) preparing a test solution by mixing 1 part by weight of a Pelargonium sidoides extract with 4) to 60 parts by weight of a 15 to 30 wt% aqueous ethanol solution, 6 to 9 parts by weight of Folin Ciocalteus phenol reagent, 4) to 60 parts by weight of a 10 to 20 wt% aqueous solution of sodium carbonate, and 15 to 4) parts by weight of water, centrifuging the mixture solution, and taking the supernatant as the test solution;(B) preparing a standard solution by mixing 1 part by weight of an aqueous ethanol solution of epicatechin with 8 to 12 parts by weight of a 15 to 33 wt% aqueous ethanol solution, 1 to 2 parts by weight of Folin Ciocalteus phenol reagent, 8 to 12 parts by weight of a 10 to 20 wt% aqueous solution of sodium carbonate, and 3 to 8 parts by weight of water, centrifuging the mixture solution, and taking the supernatant as the standard solution;(C) measuring the absorbance of the test solution and the standard solution, while using water as a control; and(D) calculating the total phenol content in terms of epicatechin by the following math figure 1 :

[Math Figure 1]

E_sx V x 200 ^{X 1 O}° wherein Et is the absorbance of the test solution,

Es is the absorbance of the standard solution,

V is the volume of the test solution measured in m-6, and

S is the mass of the standard solution measured in mg. [2] The quantitative analysis method for Pelargonium sidoides extract according to claim 1, further comprising a step of leaving the mixture solution to stand at normal temperature for 10 to 30 minutes before centrifuging the mixture solution. [3] The quantitative analysis method for Pelargonium sidoides extract according to claim 1 or 2, wherein the centrifuging is performed for 5 to 20 minutes. [4] The quantitative analysis method for Pelargonium sidoides extract according to claim 1 or 2, wherein the absorbance is measured at 720 nm. [5] The quantitative analysis method for Pelargonium sidoides extract according to claim 1 or 2, wherein the 15 to 30 wt% aqueous ethanol solution is prepared by diluting a 95 to 96 wt% aqueous ethanol solution with water at a volume ratio of 1:2 to 5.