KR100255278B1 - Natural chemical substance isolated from indian plants - Google Patents

Abstract

PURPOSE: A natural material extracted from indian plants is provided, which shows inhibition effect on growth of toxic bacteria in the intestines. Especially, a natural material extracted from Aegle marmelos shows inhibition effect on growth of Cl. perfringens. CONSTITUTION: A process for the preparation of natural material extracted from indian plants comprises the steps of: collecting 50kinds of indian plants, and preparing leaves, branch, root, flower, fruits, seeds, seed oil, oil dregs and whole plant from the indian plants; drying the plant preparation in the shade, and grinding into fine powder using a blender; extracting each sample at room temperature using methanol for 2times, and filtering; concentrating the filtrate in vacuumed condition at 35deg.C using a rotary vacuumed evaporator; testing inhibition effect on growth of bacteria in the intestines by impregnated paper disc method; and finding the fact that the natural material extracted from Aegle marmelos shows inhibition effect on growth of Cl. perfringens.

Description

Natural chemical substance isolated from indian plants

The present invention relates to natural substances extracted from tropical plants that exhibit resistance to human intestinal bacteria. More specifically, the present invention relates to the selective growth inhibitory activity of natural substances extracted from tropical plants against five enteric bacteria investigated using the impregnated paper disc method.

Various microorganisms exist as highly complex ecosystems with many species changes in the human intestinal system. It is well known that microorganisms are involved in the development of various disease states by modifying harmful or useful derivatives ingested or biologically modifying various compounds formed internally, as well as normal physiological actions. Thus, modification of these biological substances affects drug efficacy, toxicity, carcinogenesis and aging (Hentges, 1983; Mitsuoka, 1992). There were some differences in intestinal bacterial composition between patients and healthy people, and between young and older people. Normal gastrointestinal microbiota were found to be composed of Lactic acid bacteria that seem to play a large role in host defense against metabolism, infection, aging and immune-enhancing activities (Hentges, 1983; Modler et al., 1990; Mitsuoka, 1992). ).

In contrast, the microbiota composition of cancer patients or patients without Alzheimer's disease is known to consist mainly of high concentrations of Clostridia and Eubacteria with some Lactic acid bacteria (Finegold et al., 1975; Mastromarino et al., 1978; Fujisawa et. al., 1992). It has also been reported that older people have fewer Bifidobacteria but younger Clostridia than younger ones (Gorbach, 1967; Mitsoka, 1992). Microbiota disturbances can cause a variety of diseases and abnormal physiological conditions.

Recently, attention has been focused on plant-derived bifidus urea and growth inhibitors against harmful bacteria such as Clostridium perfringens, Cl.difficile and Escherichia coli. Because plants are in fact a rich source of biologically active chemicals (Namba, 1986; Rowe, 1989; Harborne, 1993; Kinghorn and Balandrin, 1993). We have reported the growth response of human enteric bacteria to oriental medicinal plant extracts (Ahn et al., 1990a, b, c; Ahn et al., 1991; Ahn et al., 1994). However, the effect of tropical plant extracts on the growth of gut bacteria has not been fully investigated despite their good pharmacological action (Grainge and Ahmed, 1988; Rowe, 1989; Umrao, 1990; Schmelzer, 1991; Kinghorn and Balandrin, 1993). ). We evaluated the growth inhibition response of human intestinal bacteria to methanol extracts of tropical plant species collected from India and Niger.

Accordingly, the present invention provides a natural material extracted from a tropical plant exhibiting resistance to human intestinal bacteria by extracting natural substances from tropical plants and conducting growth inhibition response tests of five human enteric bacteria. For that purpose.

Hereinafter, the configuration and operation of the present invention.

The present invention consists of a process of collecting plant material and using it to prepare a sample and a process of microbiologically quantifying the prepared sample material.

The five human intestinal bacterial strains used in the present invention are as follows.

Bifidobacterium bifidium (ATCC 29521), Bif. longum (ATCC 15707), Lactobacillus acidophilus (KCTC 3145), Clostridium perfringens (ATCC 13124) and Escherich ia coli (ATCC 11775).

The stock culture of these five strains is routinely stored in Eggerth-Gagnon Liver Extract-Field Slant (Mitsuoka et al., 1965) at -60 ° C. And, if necessary, secondary culture in Eggerth-Gagnon (EG) agar medium (Eiken Chemical, Japan). Plates were incubated anaerobicly for 48 hours at 37 ° C. in 5% H ₂ , 15% CO ₂ , 80% N ₂ in Glove box (Coy Lab., Michigan, USA). The next day the bacteria were grown in EG culture medium (pH 6.8).

Hereinafter, the specific configuration and operation of the present invention will be described in detail by way of examples.

Example 1 Plant Material Collection and Sample Preparation Using the Same

A total of 84 samples of 50 Indian plants from the genus 31 were selected anecdotally and are shown in Table 1 below. Various tissues: leaf (29), stem (24), root (1), flower (1), fruit (3), seed (5), seed oil (1), oily debris (1) And whole plants (19) were collected in March-April, August-September 1993 in Karnataka, India.

In addition, 27 African plant specimens (12 to 20 plant species) consisting of leaves (9), stems (4), seeds (2), fruits (1) and whole plants (11) were found in 1994 in Niger, West Africa. Collected in October. The economic significance of most of these plants has been explained in detail in other cases (Grainge and Ahmed, 1988; Umrao et al., 1990; Schmelzer, 1991).

Plant preparations were dried in the shade and finely powdered using a blender. Each sample (50 g) was extracted twice with 200 mL methanol at room temperature and filtered (Toyo filter paper No. 2, Japan).

The filtrate was concentrated in vacuo at 35 ° C. using a rotary vacuum evaporator. The yield of each sample extract is shown in Table 1 and Table 2 below.

Yield of Indian Plants Tested and Each Sample Extract Plant species Genus Sample tissue ^a yield(%) Adhatoda vasica Acanthaceae L 28 St 27 Agave americana Agavaceae Wp 5 Achyranthes aspera Amaranthaceae Wp 15 St 13 Annona squamosa Annonaceae Se 13 Nerium indicum Apocynaceae R 15 St 21 L 14 Thevetia peruviana L 20 Acorus calamus Araceae Wp 20 Calotropis gigantea Asclepiadaceae L 23 Eupatorium odoratum Asteraceae Wp 11 Eupatorium triplinerve Wp 18 Parthenium hysterophorus Wp 19 Bignonia anguiscati Bignoniaceae L 24 St 20 Opuntia elatior Cactaceae L 9 Cassia auriculata Caesalpiniaceae L 13 St 17 Cassia tora Wp 28 Cannabis sativa Cannabinaceae Wp 22 Artemisia maritima Compositae Wp 11 Acanthospermum hispidum Wp 9 Cuscuta reflexa Convolvuaceae Wp 6 Thuha occidentalis Cupressaceae L 22 St 13 Acalypha indica Equphorbiaceae L 23 Ricinus communis Euphoribiaceae L 22 Jatropha integerrima Wp 15

(continued)

Plant species Genus Sample tissue ^a yield(%) Cymbopogon citratus Garmineae Wp 19 Ocimum americanum Labiatae L 21 Wp 15 Se 14 St 13 Ocimum basilicum Wp 19 Ocimum sanctum Wp 15 Azadirachta indica Meliaceae L 26 Se 17 St 23 Melia azedarach L 21 St 17 St 24 Swietenia mahagoni L 19 Fr 20 Acacia ferruginea Mimosaceae L 18 St 17 Prosopis chinensis L 21 St 22 Ficus elastica Moraceae L 13 St 16 Bougainvillea spectabilis Nyctaginaceae L 24 St 24 Arachis hypogaea Papilionaceae Wp 23 Pongamia pinnata L 15 Se 20 St 11 Sesbania grandiflora L 22 St 22 Ziziphus mauritiana Rhamnaceae L 23

(continued)

Plant species Genus Sample tissue ^a yield(%) Aegle marmelos Rutaceae Fr 19 L 25 St 24 Murraya koenigii L 25 St 28 Bassia latifolia Sapotaceae L 15 Madhuca indica Fl 25 Oc 12 L 15 So 26 Se 23 St 15 Veronica anagallis Scrophulariaceae St 20 Datura metel var. alba Solanaceae Fr 20 L 26 St 23 St 25 Strychnos nux-vomica Strychnaceae L 19 Clerodendrum inerme Verbenaceae Wp 26 Lantana camara L 19 Lantana camara aculeata St 16 Stachytarpheta indica Wp 22 Vitex negundo L 23 St 25 Note: ^a L, leaf; Wp, whole plant; St, stem; R, root; Se, seed; Fl, flower; Fr, fruit; Oc, oily dregs; So, seed oil

Yield of tested African plants and each sample extract Plant species Genus Sample tissue ^a yield(%) Amaranthus viridis Amaranthaceae Wp 27 Blepharis linarifolia Wp 20 Celosia trigyna Wp 32 Calotropis gigantea Asclepiadaceae L 25 Cassia mimosoides Caesalpinaceae L 22 Cassia occidentalis Wp 25 Cassia tora Wp 29 Boscia senagalensis Capparidaceae L 29 St 19 Clome viscosa Wp 22 Combretum glutinotum Combretaceae L 20 St 19 Combretum micronthum Wp 26 Guiera senagalensis L 26 Se 19 Piloitigma vetilicolin Wp 27 Ipomoea asarifolia Convolvulaceae Wp 26 Azadirachta indica Meliaceae L 26 Porsopis chinensis Mimosaceae Fr 20 L 21 St 23 Bougainvillea spectabilis Nyctaginaceae L 24 Se 22 Boirerio radiata Rubiaceae Wp 19 Waltheria indica Sterculiaceae Wp 29 Balanites aegyptiaca Zygophyllaceae L 21 St 19 Note: ^a Same as described in Table 1.

Example 2. Microbiological Analysis

The impregnated paper disc method was used to analyze the effect of test specimens on growth inhibition of the intestinal bacteria used (Ahn et al., 1990).

An important factor in screening biologically active materials is the starting concentration. The plant extract of 10-20 mg / disc did not cause any problems with solubility and allowed detection of minimally active compounds (Ahn et al., 1990a, c), so that the concentration of the extract extracted in Example 1 in the present invention Was performed at 10-20 mg / disc.

One loop of bacteria was suspended in 1 mL physiological sterile water, and then 0.1 mL of the bacterial suspension was inoculated onto EG agar medium. A 10 mg sample dissolved in 100 mL of methanol was applied to a paper disc (Advantec diam. 8 mm, Toyo) using a syringe. After evaporating the solvent, the paper disc was placed on the surface of the EG agar medium in which the test bacteria were cultured. All plates were incubated anaerobicly at 37 ° C. for 2 days. All inhibition experiments were repeated three times. Growth response of the five bacterial strains to the test sample was determined in comparison to the control. At this time, the inhibition response is strong response +++, zone diameter> 20mm; moderate ++, zone diameter 10-20 mm; weak +, zone diameter 10-15 mm; Little or no response, classified as zone diameter <10mm.

Growth inhibitory activity of human intestinal bacteria on extracts isolated from Indian plant specimens is shown in Table 3 below. In addition, the experimental results performed with lactic acid bacteria are also shown in Table 3 below.

Growth Inhibitory Activity of Indian Plant Specimens Against Intestinal Bacteria Botanical name ^a part ^b Active ^c Bif.bifidum Bif.longum Lact.acidophilus Cl.perfringens E.coli A.indica L · · · + · A.hypogaea Wp · · +++ · · A.indica St · · +++ · · C.inerme Wp · · ++ · · C.citratus Wp · · · ++ · D.metel L + · · · · E.odoratum Wp · · · + · E.triplinerve Wp · · · + · N.indicum L · + · · · N.indicum R · + · · · O.americanum St · · · + · O.americanum Se · · · + · O.basilicum Wp · · · ++ ++ O.sanctum Wp · ++ ++ ++ · P.pinnata L · + · · · P.chinensis L +++ ++ +++ ++ + R.communis L · + · + · T.occidentalis L · + · · · V.anagallis St · + · · · A.ferruginea L · + · · · A.marmelos L · · · +++ · A.marmelos St · + · + + S.grandiflora L · · + · · S.grandiflora St · + ++ · · S.indica Wp · + · · · A.aspera Wp · · + · · A.hispidum Wp · · + + · M.indica Fl · · · +++ · A.marmelos Fr · ++ · + · Note: ^a plant species exhibiting activity. ^b As described in Table 1. ^c strong response +++, zone diameter>20mm; moderate ++, zone diameter 10-20 mm; weak +, zone diameter 10-15 mm; little or no response, zone diameter 〈10mm

Growth inhibition effects varied with plant species, sample tissues and bacterial strains. Of the 84 Indian plant specimens, 29 exhibited inhibitory activity. Extracts isolated from the leaves of prosopis chinensis showed the predominance of Bif. inhibited the growth of bifidm (+++). Bif, the predominant bacterium in the intestines of adults. Normal growth inhibitory activity (++) on longum was obtained from the leaves of Ocimum sanctum plants, Aegle marmelos fruit and leaves and stems of P. chinensis.

Lact. For acidophilus, extracts isolated from Arachis hypogaea whole plants, Azadirachta indica stems and P. chinensis leaves strongly inhibited the growth of these bacteria (+++), while Clerod endrum inerme whole plants, O. sanctum whole plants and Sesbania Normal inhibitory (++) activity was obtained from the extracts of the grandiflora stems.

Extracts isolated from A. marmelos leaves and Madhuca indica flowers were Cl. It showed strong inhibitory activity against perfringens, whereas it showed moderate inhibitory activity from extracts of whole plants of Cymbopogon citratus, Ocimum basilicum, O. sanctum and P. chinensis leaves. In experiments with E. coli, only extracts isolated from whole plants of O. basilicum showed normal growth inhibition. Growth inhibitory activity of African plant specimens against the tested enteric bacteria is shown in Table 4 below.

Growth Inhibitory Activity of African Plant Specimens Against Intestinal Bacteria Botanical name ^a part ^b Active ^c Bif. bifidum Bif. longum Lact. acidophilus Cl. perfringens E. coli C. mimosoides L · + · · · G.senagalensis L · + · · · G. senagalensis S · + · · · B. radiata Wp · + · · · A. viridis Wp · + · · · C. tora Wp · ++ · · · P. chinensis L +++ ++ +++ ++ + P. chinensis St · · · + · Note: a indicates plant species exhibiting activity. B Description shown in Table 1. c Description shown in Table 3.

As a result, growth responses similar to Indian plant specimens were observed from African plant specimens. Like the leaves of Indian P. chinensis, African species extracts also contain lactic acid bacteria and Cl. Each showed strong and moderate growth inhibition for perfringens. Cassia tora Extract of the entire plant is Bif. Moderate growth inhibition was shown for longum. Other plant specimens showed little or no activity with respect to the organisms used.

As described through the above Examples and Experimental Examples, the present invention has the effect of providing a growth inhibitory use of the human intestinal bacteria of natural substances extracted from tropical plants. Furthermore, it can be seen from the present invention that the harmful bacteria are larger than the lactic acid bacteria in the growth inhibitory effect of the human intestinal bacteria.

In particular, the material extracted from Aegle marmelos is Cl. Obvious growth inhibitory effects have been identified on perfringens, and it can be added to beverages or foods to obtain effective effects of inhibiting the growth of human intestinal bacteria.

Claims (1)

Aegel marmelos A natural substance that inhibits the activity of harmful intestinal bacteria in humans, which is extracted and separated from the fruit.