TWI820946B - Oral pharmaceutical composition with anti-periodontal bacteria adhesion function - Google Patents

Abstract

The present invention provides a pharmaceutical composition for oral health care, particularly an oral pharmaceutical composition with the function of resisting periodontal bacterial adhesion. The pharmaceutical composition contains chlorella extract, Phellinus linteum polysaccharide extract and borage oil extract. It is available as a tablet and prevents periodontal bacteria biofilm from adhering to teeth and gums. The chlorella extract in the oral pharmaceutical composition contains a variety of short-chain peptides and chlorella growth factor (CGF), which can promote the normal development of teeth and bones and quickly change the oral flora ecology. Phellinus linteus polysaccharide The extract contains a high concentration of Phellinus linteus polysaccharide (β-1,3/1,6 glucan), while the borage oil extract is rich in natural polyunsaturated fatty acids Omega-6, which has the functions of promoting body fluids and quenching thirst, The effect of annealing and reducing fire.

Description

Oral pharmaceutical composition with anti-periodontal bacteria adhesion function

The present invention relates to a pharmaceutical composition that can be used as oral health care, and in particular to an oral pharmaceutical composition that has the function of preventing periodontal bacteria from adhering to teeth and gums.

Periodontal bacteria have been identified as one of the main causes of periodontal disease. According to a survey by the National Health Service, 99.2% of the population in Taiwan suffers from varying degrees of periodontal disease, which means that less than 200,000 people in Taiwan have healthy periodontal disease. If periodontal health continues to deteriorate, it may lead to loosening and loss of teeth, causing periodontal inflammation or increasing the chance of periodontal inflammation. Current treatment methods for periodontal disease mainly include manual removal of surface calculus by doctors, surgical root planing (Scaling and root planning, SRP), or oral medications. However, long-term use of drugs can cause oral bacteria to become drug-resistant and may cause unknown side effects to health. According to the health education information from the Periodontology Department of the Wing General Hospital in Taichung, the main cause of periodontal disease is that bacteria in the mouth adhere to the surface of the teeth, between the teeth, and on the edges of the gums to form dental plaque. If not properly cleaned, , will cause persistent inflammation in the oral cavity, destroy periodontal tissue, and form periodontal disease; the health education materials of Mackay Memorial Hospital point out that periodontal disease is a disease of the supporting tissues around the teeth, such as gums and alveolar bones. , the early stage of periodontal disease is also called "gingivitis". Symptoms include red and swollen gums, easy gum bleeding when brushing, bad breath, gum recession, etc., but it has not yet caused damage to the alveolar bone; while the advanced stage of periodontal disease is called "gingivitis". "Periodontitis", in addition to the worsening of the symptoms of gingivitis mentioned above, symptoms such as tooth shaking, gum suppuration, and tooth position deviation may also occur.

The latest research from the European Federation of Periodontology in the latest issue of "Journal of Clinical Periodontology" shows that if COVID-19 patients also suffer from periodontal disease, the proportion of severe cases requiring the use of respirators is 3.67 times higher, and the mortality rate is 3.67 times higher. It is 9 times higher. Researchers analyzed 568 patients with confirmed COVID-19 and found that 33 of 258 patients with periodontitis developed Covid-19 complications, but only 7 of 310 patients without periodontitis. complications, study It is pointed out that COVID-19 is related to a potentially fatal inflammatory reaction, and patients with periodontal disease are 3.67 times more likely to develop complications after being infected with COVID-19. Shapira Lior, President of the European Federation of Periodontology, also pointed out: "The research results show that periodontal disease may be a factor that makes the new coronavirus more severe. Therefore, if periodontal disease is related to the condition of COVID-19 patients, There is a cause-and-effect relationship between aggravation, so maintaining periodontal health requires special attention during treatment.”

In addition, Teughels et al. previously disclosed that a composition of a specific beneficial bacterial strain, Lactobacillus reuteri , can be used to improve the oral flora of patients with chronic periodontal disease [ J. of Clinical Periodontology , 40 (2013), 1025 -1035]. Based on clinical results, the authors found that the administration of this beneficial bacterial strain has clinically relevant benefits for patients. This is because when Lactobacillus loderi oral tablets are used as an adjuvant for scaling and root planing procedures and oral hygiene instructions, " The outcome measures of "risk of disease progression" and "need for additional surgery" were significantly better, indicating that oral lozenges are a better strategy in selecting the dosage form of periodontal care pharmaceutical compositions.

In addition, relevant researchers all hope to develop ingredients from natural plants that can resist periodontal disease bacteria or prevent periodontal disease bacteria from adhering to teeth, so as to avoid the side effects of common Western medicines. However, current research in this area focuses on the development of Chinese herbal medicines, and through fermentation technology, the components of Chinese herbal medicines are metabolized by microorganisms into functional small molecules, which is beneficial to the human body to exert their effects; however, when it comes to mass production of Chinese herbal medicines, , most of them are commercialized in the powder form of scientific Chinese medicine, which must be taken orally to exert its effect in the human body. However, the taste of Chinese herbal medicine is not universally accepted by the public, and it is not easy for most people to take it for a long time, and in terms of oral health care It is said that the oral administration method cannot stay in the mouth for too long, which will reduce its effectiveness on the human body. Therefore, if a new generation of oral health care products in the form of natural oral tablets that do not contain Western medicine or antibiotics can be developed, it will be of great help to the progress of oral medicine in my country.

In view of the aforementioned side effects or medication adaptability caused by the conventional use of Western medicine or traditional Chinese medicine to treat periodontal disease, and to further overcome the limited action time of general oral medicines in the oral cavity, one of the purposes of the present invention is to provide a method that has less side effects and An oral pharmaceutical composition containing natural plant ingredients. Another purpose of the present invention is to extend the action time of the pharmaceutical composition through oral tablet form, so that the adhesion of periodontal disease bacteria to teeth and gums can be reduced, thereby preventing or Treat the onset of periodontal disease.

The embodiment of the present invention provides an oral pharmaceutical composition with the function of resisting the adhesion of periodontal bacteria, including chlorella extract, Phellinus linteus polysaccharide extract and borage oil extract. The oral pharmaceutical composition is for oral administration. Lozenge dosage form.

In one embodiment of the present invention, the weight percentages of the chlorella extract, the Phellinus linteus polysaccharide extract and the borage oil extract are respectively about 36%~40%, 36%~40% and 20%~ 28%.

In an embodiment of the present invention, the weight percentages of the chlorella extract, the Phellinus linteus polysaccharide extract and the borage oil extract are approximately 38%, 38% and 24% respectively.

In another embodiment of the present invention, the minimum dose of the chlorella extract in the oral pharmaceutical composition can be about 40 to 45 mg/ml, preferably about 42 mg/ml.

In another embodiment of the present invention, the oral pharmaceutical composition further includes an excipient.

In yet another embodiment of the present invention, the oral pharmaceutical composition can reduce the adhesion of Porphyromonas gingivalis .

In yet another embodiment of the present invention, the oral pharmaceutical composition can reduce the adhesion of Streptococcus sp .

In another embodiment of the present invention, the chlorella extract in the aforementioned oral pharmaceutical composition can be extracted with hot water. The extract contains chlorella short-chain active ingredients such as chlorella protein peptides and green algae growth factors. Peptides, and the molecular weight of these peptides can range from about 1KDa to 30KDa.

On the other hand, in one embodiment of the present invention, the Phellinus linteus polysaccharide extract can be extracted with hot water and contains about 40% β-1,3/1,6 glucan; and the borage oil The extract contains approximately 15% gamma-linolenic acid.

The oral pharmaceutical composition provided by the present invention contains active ingredients in natural products such as chlorella, Phellinus linata and borage, which can resist periodontal disease bacterial flora biofilm adhesion to teeth and gums, and is prepared as an oral composition. The tablet form will be convenient for ordinary people to use in daily life. In addition to the aforementioned inconvenience of usage and possible adverse side effects when using western medicine and antibiotics.

The embodiments of the present invention will be further described below. The examples listed below are used to illustrate the present invention and are not intended to limit the scope of the present invention. Anyone familiar with this art can, without departing from the spirit and scope of the present invention, Some modifications and modifications may be made, so the scope of protection of the present invention shall be determined by the appended patent application scope.

Figure 1 shows the colony inhibition results of Porphyromonas gingivalis (17689 Porphyromonas gingivalis ) co-cultured with the test substance.

Figure 2 shows the colony inhibition results of Streptococcus genus (15977 Streptococcus sp. ) co-cultured with the test substance.

Figure 3 shows the results of inhibiting colony biofilm attachment by co-culture of Porphyromonas gingivalis (17689 Porphyromonas gingivalis ) and the test substance.

Figure 4 shows the results of inhibition of colony biofilm attachment by co-culture of Streptococcus genus (15977 Streptococcus sp .) and the test substance.

Figure 5 shows the test results of different tested substances in the animal model of periodontal disease in SD rats; (A) is the blank control group, (B) is the chlorella extract group, and (C) is the sulfahoudin group.

Example 1 Preparation of components of oral pharmaceutical composition

Example 1.1 Preparation of Chlorella Extract

The source of chlorella is Chlorella vulgaris . First, put the Erlenmeyer flask into the prepared culture medium, and sterilize it in a sterilizing kettle at 121°C for 15 minutes. After cooling, start pouring. Before pouring, irradiate the blank plate with UV and let it dry for 30 minutes. The initial concentration of Chlorella in algae plate culture is 0.01g/100ml (about 6x10 ⁷ algal cells), that is, add 100ml RO water and 15g/L agar to prepare 100ml culture medium, and wait until it is cultured to 0.5g/100ml (about 3x10 ⁹ cells) Algae cells), centrifugally filter to obtain algae mud and dry it, take 100g of chlorella powder, add liquid nitrogen to destroy the cell wall, and dissolve it with 3000ml of deionized water. Then use an autoclave to extract for 20 minutes (121°C, 1.2kg/cm ² ). Repeat the above steps three times. After the suspension is cooled, centrifuge (8800g, 20 minutes) to collect the supernatant, and then add the supernatant to the stomach. The protease performs an enzymatic hydrolysis reaction, and after the reaction is completed, it is precipitated with four times the volume of 95% alcohol. The precipitate is washed twice with an equal volume of 75% alcohol, and finally the precipitate is freeze-dried to obtain a chlorella extract containing chlorella protein peptides and green algae growth factors with a peptide sequence of about 1~30kDa.

Example 1.2 Preparation of Phellinus linteus polysaccharide extract

Place 10g of the commercially available fruiting bodies of Phellinus linteus in an Erlenmeyer flask containing 100 ml of deionized water, bathe in a 90°C water bath for 2 hours, use filter paper to suction and filter, and extract the filter residue twice under the same conditions. After collecting twice, a filtrate containing approximately 40% of Phellinus linteus mycelium polysaccharides [containing approximately 10% of β-1,3/1,6 glucan] can be obtained. Concentrate it under reduced pressure to 20 ml, use freeze drying to remove water, weigh the required dose of powder, redissolve it with an appropriate solvent, and store it at -20°C for later use.

Example 1.3 Preparation of Borage Oil Extract

Borage oil extract can be obtained from commercially available borage oil obtained by pressing or low-temperature extraction of borage ( Borago officinalis ) seeds. It contains about 15% polyunsaturated fatty acid Omega-6 [such as γ-linolenic oil] acid (Gamma Linolenic Acid, GLA)].

Mix the chlorella extract, Phellinus linteus polysaccharide extract and borage oil prepared in the aforementioned Examples 1.1 to 1.3 in weight percentages according to the following proportions to obtain the oral pharmaceutical composition (Surahoudine) of the embodiment of the present invention. Mixture. Among them, chlorella extract accounts for about 36% to 40%, Phellinus linteus polysaccharide extract accounts for about 36% to 40%, and borage oil accounts for about 20% to 28%. Mix the three evenly and set aside. Preferably, 38% of chlorella extract, 38% of Phellinus linteus polysaccharide extract, and 24% of borage oil can be mixed evenly for later use.

Example 2 Paper tablet inhibitory concentration test (Minimum inhibitory concentration, MIC)

The periodontal disease pathogenic bacteria Porphyromonas gingivalis (purchased from the Biological Resource Conservation and Research Center of the Institute of Food Industry, No. BCRC 17689) and the pathogenic bacteria Streptococcus sp. (purchased from the Institute of Food Industry Biology Resource Conservation and Research Center, No. BCRC 15977). The aforementioned bacterial species were cultured on a culture medium plate using an appropriate culture medium. The test was divided into sterile water group, chlorella extract group, Phellinus linteus polysaccharide extract group, and Shuyahouding compound group consisting of chlorella extract, Phellinus linteus polysaccharide extract and borage oil extract. (Oral pharmaceutical composition according to the embodiment of the present invention) and a tetracyclic antibiotic group. Combine Phellinus linteus polysaccharide extract (concentration: 160mg/ml), chlorella extract (concentration: 160mg/ml), 2D water (sterile water), sulfahoudin (concentration: 160mg/ml), tetracycline After the samples to be tested (concentration of 80 mg/ml) were mixed with the culture medium, 0.6 ml was pipetted into the inoculated paper ingots and placed on the aforementioned culture medium plate. Incubate at 37°C for 24 and 48 hours, and then count the ingots. The antibacterial effect, the results are shown in Figures 1 and 2.

It can be seen from the paper tablet test results in Figure 1 and Figure 2 that although the antibacterial effect of the tetracyclic antibiotics is significant at 24 hours and 48 hours, in the Phellinus linteus polysaccharide extract group, chlorella extract, and suahoudin group, the Neither Porphyromonas gingivalis nor Streptococcus species appear to produce zones of inhibition.

Example 3 Biofilm Attachment Test

Similarly, Porphyromonas gingivalis and Streptococcus were cultured on the culture plate to form colonies. After the amount of blood plate bacteria was quantified and the quantitative colonies were scraped off with an inoculation loop, they were inoculated into sterile water (2D water) containing sterilization. Take Phellinus linteus polysaccharide extract (concentration is 10 μg/ml), chlorella extract (concentration is 10 μg/ml), 2D water (control group), sulfahoudin (concentration is 10 μg/ml), tetracycline (concentration: 10 μg/ml) was added to the culture for 24 hours, and the light transmittance was measured with a spectrophotometer at a wavelength of 490 nm, and the percentage of Porphyromonas gingivalis or Streptococcus before/after attachment (%) The results of the biofilm attachment rate of periodontal disease pathogenic bacterial colonies were analyzed. The results are shown in Figures 3 and 4 respectively.

It can be seen from the results in Figure 3 that for Porphyromonas gingivalis, the adhesion rate of the suahoudin group was 42%, which was better than that of the chlorella extract group (adhesion rate of 56%) and Phellinus phyllus polysaccharide extract group ( 53%). It can be seen from Figure 4 that for Streptococcus genus, the adhesion rate of the surahoudine group of 39% is also significantly better than that of the chlorella extract group (adhesion rate of 46%) and Phellinus linteus polysaccharide extract group (47 %), and the adhesion rates of all groups were lower than the 2D water control group and higher than the tetracycline control group, indicating that the oral pharmaceutical compositions of the embodiments of the present invention (Surahoudine group) can reduce periodontal disease. Although the pathogenic bacteria colony biofilm is attached, it is not as effective as the antibiotic group, but it still has obvious effects. It is a very successful test result on the premise of avoiding the use of antibiotics to treat antibiotics that may lead to the development of drug resistance. On the other hand, after testing, the embodiments of the present invention The minimum effective concentration of chlorella extract in the oral pharmaceutical composition can be about 40~45 mg/ml, preferably about 42 mg/ml.

Example 4 Animal model test of periodontal disease in rats

In this embodiment, the animal test was conducted using a Ligature-induced periodontal disease model in rats. The rats used in the experiment were male rats of the Sprague Dawley (SD) strain, weighing approximately 280 to 310 grams. The animals are sourced from the National Animal Center. The environment temperature is 23~25℃, the light cycle is controlled at 12 hours each day and night (AM8:00-PM8:00), and the animals are kept in an animal room with appropriate humidity and independently filtered air supply. And adequate supply of feed and drinking water. After the mice were anesthetized with Hydrocholine (1ml/0.1kg) by intraperitoneal injection, the ligation line was placed on the neck of the first molar on both sides of the lower jaw and the second largest molar on the upper jaw, and the knot was placed on the buccal side. After tying the mycelium thread mixed with the aforementioned Porphyromonas gingivalis or Streptococcus bacterial fluid on the heart surface, immediately loosen the thread and apply hydrogel (Polyethyleneglycol, PEG) to the teeth/gum. At the same time, after the operation, the hydrogel blank group, the hydrogel plus 10 μg/ml chlorella extract group, the hydrogel plus 10 μg/ml sulfahoudine group, and other groups were applied to the oral cavity. The experimental animals in each group were The SD rats in the silk thread-induced control group were sacrificed on the 28th day after periodontal disease developed, and their tissues were taken to observe the periodontal disease status. The results are shown in Figure 5.

Figure 5(A) shows that on the tissue of the hydrogel blank group without any test substance being given a normal diet, it can be seen that there are many small pustules in the gums of the test animal SD rats, while in Figure 5(B) the hydrogel In the group given with chlorella extract, it can be seen that the pustules in the gums of the test animal SD rats have shrunk. In the group given the hydrogel plus Shuahoudin in Figure 5(C), it can be seen that the test Animals: No pustules can be seen in the gums of SD rats. It was shown that the oral pharmaceutical composition (Surahoudine group) according to the embodiment of the present invention can prevent the biofilm attachment of periodontal disease pathogenic bacterial colonies and prevent the oral inflammation caused by periodontal disease in the test SD rats.

Oral pharmaceutical compositions according to embodiments of the present invention may further include pharmaceutically acceptable carriers that are well known to those skilled in the art. These carriers may include but are not limited to: solvents, buffers, emulsifiers, Suspending agents, disintegrants, dispersants, decomposing agents, binders, stabilizers, excipients, preservatives, diluents, liposomes, sweeteners, flavoring agents, dyeing reagents and similar ingredients. The carrier may be composed of one or more than two of the aforementioned components.

Claims (10)

An oral pharmaceutical composition with the function of resisting the adhesion of periodontal bacteria. The oral pharmaceutical composition includes a Chlorella extract, a Phellinus mori polysaccharide extract and a Borage oil extract. The oral pharmaceutical composition is in one mouthful. Tablet-containing dosage form; wherein, the chlorella extract is prepared by taking chlorella powder, destroying the cell wall of chlorella, extracting it with hot water, and then performing an enzymatic hydrolysis reaction with pepsin; the Phellinus linteus polysaccharide extract The borage oil extract is obtained by extracting the fruiting bodies of Phellinus linata with hot water; and the borage oil extract is obtained by squeezing the borage seeds. The oral pharmaceutical composition with anti-periodontal bacteria adhesion function as described in claim 1, wherein the weight percentages of the chlorella extract, the Phellinus linteus polysaccharide extract and the borage oil extract are respectively about 36%~ 40%, 36%~40% and 20%~28%. The oral pharmaceutical composition with anti-periodontal bacteria adhesion function as described in claim 2, wherein the weight percentages of the chlorella extract, the Phellinus linteus polysaccharide extract and the borage oil extract are respectively about 38%, 38% and 24%. The oral pharmaceutical composition with anti-periodontal bacteria adhesion function as described in claim 2, wherein the minimum dose of the chlorella extract is about 42 mg/ml. The oral pharmaceutical composition with anti-periodontal bacteria adhesion function as claimed in claim 2, wherein the oral pharmaceutical composition further contains an excipient. The oral pharmaceutical composition with anti-adhesion function of periodontal bacteria as described in claim 1, 2 or 3, wherein the periodontal bacteria is Porphyromonas gingivalis . The oral pharmaceutical composition with anti-adhesion function of periodontal bacteria as described in claim 1, 2 or 3, wherein the periodontal bacteria is Streptococcus sp . The oral pharmaceutical composition with anti-periodontal bacteria adhesion function as described in claim 1, 2 or 3, wherein the chlorella extract contains chlorella short chains such as chlorella protein peptides and green algae growth factors. Active peptides, and the molecular weight of these peptides is between about 1KDa and 30KDa. The oral pharmaceutical composition with anti-periodontal bacteria adhesion function as described in claim 1, 2 or 3, wherein the Phellinus linteus polysaccharide extract contains about 40% β-1,3/1,6 glucan polysaccharide. . The oral pharmaceutical composition with anti-periodontal bacteria adhesion function as described in claim 1, 2 or 3, wherein the borage oil extract contains about 15% γ-linolenic acid.