WO2024013586A1

WO2024013586A1 - Injectable herbal ointment for the treatment of subclinical mastitis: combining spirulina algae extract and mangrove leaf extract

Info

Publication number: WO2024013586A1
Application number: PCT/IB2023/056329
Authority: WO
Inventors: Sadaf SHIRANI; Pargol GHAVAM MOSTAFAVI; Ali MASHINCHIAN MORADI; Rezvan ATTARI
Original assignee: Shirani Sadaf; Ghavam Mostafavi Pargol; Mash Moradi Ali
Priority date: 2023-06-19
Filing date: 2023-06-19
Publication date: 2024-01-18

Abstract

the process for preparing an injectable herbal ointment for the treatment of subclinical mastitis, comprising: Combining 300 mg of ethanolic extract of spirulina algae (Platensis Spirulina) and 90 mg of ethanolic extract of mangrove leaves (Avicennia marina) in each 10 ml tube of the ointment and adding 10 ml of sanitary liquid paraffin as a carrier. the extraction process is carried out using the ethanol method with a 10% mass-volume immersion technique. the extracts are sterilized using a 0.22-micron microbial filter. The ointment is supplied as an intramammary ointment for the treatment of subclinical mastitis.

Description

Injectable Herbal Ointment for the Treatment of Subclinical Mastitis: Combining Spirulina Algae Extract and Mangrove Leaf Extract

A61Q 19/00 – A61K 36/00

Mastitis is characterized by the inflammation of parenchymal tissue in the mammary glands, accompanied by an increase in somatic cells as an indicator of the disease (scc > 400,000). This inflammation is typically caused by impact or infection from microorganisms and bacteria. Subclinical mastitis is the most significant type of mastitis and is considered one of the costliest diseases affecting dairy cows worldwide. It leads to reduced milk quality, increased treatment costs, higher abortion rates, decreased lactation, diminished fertility in cows, increased microbial load in milk, and alterations in milk composition such as reduced lactose, casein, fat, vitamins, and minerals (e.g., phosphorus, potassium, calcium). Furthermore, raw milk undergoes enzyme changes that result in the loss of nutritional value. Severe cases may require the removal of affected animals.

The most common method of treating mastitis in cattle is through intra-mammary injection of antibiotics. Approximately 80% of antibiotic residues in milk are attributed to mastitis treatments, both during the lactation period and the dry period of cows. The presence of pharmaceutical residues in milk is a significant concern for food safety. Antibiotics in milk can interfere with the production of dairy products, such as yogurt and cheese, which are produced through fermentation. Moreover, they can contribute to antibiotic resistance in various bacterial species and cause milk leakage during antibiotic treatment.

Recent advancements in the treatment of mastitis include:

Control of mastitis infection in dairy cows using Ag/Zno nanoparticles (Registration Number: 88993): This invention aims to control mastitis in dairy cows through intramammary injection of Ag/Zno nanoparticles at two doses, 10 times Mbc (Minimum Bacterial Concentration) and 15 times Mbc. Staphylococcus aureus, Escherichia coli, and Streptococcus agalactiae bacteria were introduced to 138 cows, resulting in reduced somatic cell counts and the efficacy of Ag/Zno in controlling mastitis-causing bacteria. The wide spectrum of nanoparticles used in this invention exhibits unique properties compared to common antibiotics. However, the compounds and herbal ointment described in this invention are entirely different from the present invention for the treatment of subclinical mastitis in dairy cows.

Use of medicinal herbs, including henna, rosemary, chamomile, peppermint, and bay leaves, for mastitis treatment in cows (Registration Number: 92888): This invention involves a herbal combination of henna, rosemary, mint, bay leaves, and chamomile in specific percentages formulated as a mammary herbal ointment. It was tested on 50 cows with a dosage of one application in the middle of the udder for one week. The resulting herbal ointment can be used to treat clinical and subclinical mammary tumors as a weekly application in the middle of the udder. This invention does not require the use of antibiotics for mastitis treatment, and its composition and duration of administration are distinct from the present invention.

In patent number 9827284, dated 28/11/2017, by Patil, Prashant Neminath (Mumbai, IN), herbal compounds were described that enhance lactation in farm animals and reduce the occurrence of infections such as mastitis. The prescribed compounds include extracts of bioactive substances from asparagus, cotton, fennel, watercress, wheat flower, peach lotus, wind cheese, and tallow, along with a mixture of minerals and calcium diphosphate. These compounds are administered orally to livestock, improving blood circulation in the mammary glands and reducing the risk of mastitis infection. It is worth noting that this invention differs from ours, as our injectable ointment for subclinical mastitis treatment utilizes alcoholic extracts of mangrove leaves and spirulina algae.

Cream with spirulina algae

DE202016007098U1

Cream with components of spirulina algae, characterized in that these components of spirulina algae from the fresh mass of Spirulina platensis prepared and these constituents of spirulina algae are at least 1 Ma% contained in the cream.

PULSATILLA CHINENSIS EXTRACT IN PREPARING DRUG FOR TREATING VIRAL AND/OR BACTERIAL DISEASES

United States Patent Application 20220273685

Use of the compound Pulsatilla Saponin B4 represented by formula (I), or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating viral and/or bacterial diseases, and a medicament comprising the compound Pulsatilla Saponin B4 represented by formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the disease is dairy cow mastitis.

USES OF PULSATILLA CHINENSIS EXTRACT IN PREPARING DRUG FOR TREATING VIRAL AND/OR BACTERIAL DISEASES

United States Patent Application 20210038622

Antimicrobial-antibiofilm compositions and methods of use thereof

United States Patent 9936712

Compositions comprising chelating agents, metal ion salts, gelling agents or a buffer, antimicrobials, antibiofilm agents and a pH adjuster or a buffer for the prevention and treatment of wound infections and food-borne diseases involving bacterial biofilms are disclosed. The anti-infective properties of a composition include reduction or killing of anaerobic/aerobic/facultative gram-negative and gram-positive wound infection associated bacteria occurring in polymicrobial biofilms. The composition may be in the form of lotion, cream, ointment, dressing, bandage, rinse, soak, gel, spray, or other suitable forms, including certain devices. Additionally, the invention offers an efficient method of delivering the formulated composition containing one or two chelating agents or chelating agents alone or in combination with a metal ion salt using either a nanoparticle or other efficient delivery systems.

Our inventive solution can effectively control subclinical mastitis without any side effects, thanks to the use of these plant extracts. The purpose of developing this ointment is to leverage the antibacterial and anti-inflammatory properties of the alcoholic extract of mangrove leaves and spirulina algae. By reducing mastitis occurrence and minimizing the need for antibiotics in dairy cows, our ointment enhances productivity and prevents milk wastage while maintaining the quality and taste of the milk. Furthermore, our solution does not require milk circulation during usage and does not affect the milk's quality.

The mangrove plant contains various compounds, including phytoalexins and triterpenes, known for their anti-infective properties. Using this extract minimizes the risk of developing microbial resistance and treatment-resistant diseases. It also inhibits bacterial growth in milk and reduces somatic cell counts, effectively treating subclinical mastitis in cows. Extracts obtained from Spirulina platensis microalgae, which contain linolenic acid, have been recognized for their high concentration of fatty acids with antibiotic properties. These fatty acids, particularly oleic acid and palmitic acid, exhibit antimicrobial activity when combined with other fatty acids in spirulina microalgae lipids. The lipids can penetrate the bacterial cell wall's peptidoglycan network, leading to the disruption and collapse of the bacterial membrane. Phycocyanin, acting as an antibody, receptor, and biological molecule, inhibits cell proliferation through a fluorescence reaction. Similar to many microalgae polypeptides, phycocyanin binds to gene or amino acid protein sequences (plasmids, chromosomes), causing the inactivation of a group of transpeptidase catalysts involved in the final stages of peptidoglycan synthesis within the bacterial wall. This inhibition of cell wall synthesis ultimately leads to the death of bacterial cells.

Subclinical mastitis is a prevalent form of intramammary inflammation characterized by an infection in the breast accompanied by an increase in somatic cell count. Unlike acute mastitis, subclinical mastitis does not exhibit visible signs of breast tissue inflammation, and the milk appears normal. Traditional treatment approaches rely on antibiotics, which contribute to the spread of drug resistance and leave antibiotic residues in milk and meat. These issues pose economic challenges and result in significant losses. Given the problems associated with this disease, there is a clear need for an effective solution.

The claimed invention addresses this need by offering a product that combines ethanolic extracts of mangrove plants and spirulina algae, formulated with paraffin as an intramammary injection. The product contains various compounds such as alkaloids, flavonoids, glycosides, phenols, saponins, tannins, phytoalexins, steroids, and triterpenes, which possess anti-inflammatory and antimicrobial properties. Additionally, extracts extracted from spirulina platensis microalgae contain a high concentration of linolenic acid, a fatty acid known for its antibiotic properties. Other fatty acids, particularly oleic acid and palmitic acid, enhance the antimicrobial activity of spirulina microalgae lipids through synergistic effects. The lipids effectively combat bacteria, fungi, and yeast by penetrating the peptidoglycan network of the bacterial wall, leading to disruption and collapse of the bacterial membrane.

This product offers a solution for curing subclinical mastitis in dairy cows. Its use minimizes the risk of microbial resistance and the development of treatment-resistant diseases. It also inhibits bacterial growth in milk and reduces somatic cell counts, thus effectively treating subclinical mastitis in cows. By relying on natural extracts and fatty acids, this invention offers a safer and more sustainable alternative to antibiotic treatment, addressing the concerns of antibiotic resistance and the presence of antibiotic residues in dairy products.

Mastitis refers to the inflammation of parenchymal tissue in the mammary glands, characterized by an increase in somatic cells (indicated by scc > 400,000). The inflammation is typically caused by impact or infection resulting from microorganisms and bacteria. Subclinical mastitis is a significant form of mastitis, proving to be one of the costliest diseases affecting dairy cows worldwide. It leads to reduced milk quality, increased treatment expenses, heightened abortion rates, decreased lactation, diminished fertility in cows, higher microbial loads in milk, and alterations in milk composition such as reduced lactose, casein, fat, vitamins, and minerals (e.g., phosphorus, potassium, calcium). Moreover, raw milk experiences changes in enzymes that compromise its nutritional value. Severe cases may necessitate the removal of affected animals. Although antibiotic treatment is commonly used to control the disease, it contributes to the spread of drug resistance and results in the presence of antibiotics in milk and milk secretions. This invention aims to address these concerns by developing an injectable ointment for livestock that utilizes alcoholic extracts of mangrove leaves and spirulina algae.

The ointment's antibacterial and anti-inflammatory properties are expected to reduce mastitis occurrence, minimize antibiotic use in dairy cows, increase productivity, and prevent milk waste while maintaining milk quality and taste. Mangrove plants contain various compounds, including phytoalexins and triterpenes, which possess antibacterial and anti-inflammatory properties. Employing this extract minimizes the risk of developing microbial resistance and drug-resistant diseases, inhibits bacterial growth in milk, and decreases somatic cell counts in cows through subclinical mastitis treatment. Extracts derived from spirulina microalgae (specifically Spirulina platensis) are rich in linolenic acid, known for its antibiotic properties at high concentrations. Linolenic acid is considered one of the primary factors responsible for the antimicrobial activity of spirulina microalgae. Additionally, other fatty acids present in spirulina, especially oleic acid and palmitic acid, contribute to its antimicrobial effects through a synergistic mechanism. These lipids can penetrate the peptidoglycan network of the bacterial cell wall, leading to its disruption and collapse. Furthermore, phycocyanin, an antibody-like molecule, inhibits cell proliferation through a fluorescence reaction. Similar to polypeptides found in various microalgae, this molecule binds to genes or amino acid protein sequences (plasmids, chromosomes), resulting in the inactivation of a group of trans peptidase catalysts involved in the final stages of peptidoglycan synthesis within the bacterial wall. Consequently, by inhibiting cell wall synthesis, bacterial cells are eliminated.

Overall, this invention presents an injectable herbal ointment that combines spirulina algae extract and mangrove leaf extract for the treatment of subclinical mastitis in dairy cows. The ointment's antibacterial and anti-inflammatory properties have the potential to reduce mastitis cases, decrease antibiotic usage, increase milk productivity, and prevent milk waste, while maintaining the quality and taste of the milk produced.

Solution of problem

Recent advancements in the field of medicinal plants have paved the way for the prevention and treatment of various diseases in animals and humans, while minimizing the side effects associated with conventional drugs. Additionally, given the widespread issue of antibiotic resistance in livestock and the concerns surrounding antibiotic residues in milk and meat, researchers have been exploring alternative antimicrobial substances.

The present invention represents the first study demonstrating the efficacy of the intermammary administration of an alcoholic extract obtained from the mangrove plant in controlling subclinical mastitis during lactation in dairy cows. Subclinical mastitis is the most prevalent form of intermammary inflammation, characterized by increased somatic cell counts without visible signs of breast tissue inflammation or abnormal milk appearance. While antibiotic treatment is typically employed to manage acute mastitis, it contributes to the proliferation of drug-resistant microorganisms and the presence of antibiotic residues in milk and meat, resulting in economic losses.

The mangrove plant, scientifically known as Avicennia marina, belongs to a group of halophytes and salt-tolerant plants found in tidal forests and coastal regions, such as the provinces of Sistan and Baluchistan, Hormozgan, and Bushehr. This plant exhibits various chemical compounds, including alkaloids, flavonoids, glycosides, phenols, saponins, and tannins, which possess potent anti-inflammatory and antibacterial properties. Notably, the plant contains phytoalexins, steroids, and triterpenes. Phytoalexins are a diverse group of compounds, such as alkaloids and quinones, which serve as essential defense mechanisms against microbial agents. Triterpenes also exhibit antibacterial properties, with some disrupting the cell membrane to exert their effects. Moreover, both phytoalexins and triterpenes possess anti-inflammatory properties.

By utilizing the alcoholic extract of the mangrove plant, this invention mitigates the risks associated with microbial resistance, milk contamination from antibiotics, and the emergence of treatment-resistant diseases in humans. The unique properties of the plant's compounds make it a promising candidate for effectively controlling subclinical mastitis in dairy cows without the drawbacks associated with conventional antibiotic treatments.

Spirulina, scientifically known as Spirulina platensis, is a species of blue-green algae belonging to the cyanobacteria branch. Cyanobacteria are filamentous, thread-like organisms that lack heterocysts and are commonly found in shallow, alkaline areas of seas and lakes. Recent studies have demonstrated the antibacterial effects of extracellular products derived from spirulina microalgae on various bacteria.

Extracts obtained from spirulina platensis microalgae contain high concentrations of linolenic acid, which is a fatty acid with antibiotic properties. This fatty acid, along with other fatty acids such as oleic acid and palmitic acid, found in spirulina microalgae lipids, exhibits antimicrobial activity. These lipids can penetrate the peptidoglycan network of the bacterial cell wall, leading to the disruption and collapse of the bacterial membrane. This mechanism enables them to effectively eliminate bacteria, fungi, and yeast.

Another significant component of spirulina is phycocyanin, which serves as an antibody, receptor, and biological molecule. Phycocyanin prevents cell proliferation through a fluorescence reaction. Similar to polypeptides found in many microalgae, phycocyanin binds to gene or amino acid protein sequences (plasmids, chromosomes), causing the inactivation of a group of transpeptidase catalysts involved in the final stages of peptidoglycan synthesis within the bacterial wall. Consequently, by inhibiting cell wall synthesis, it leads to the death of bacterial cells.

In this study, 75 quarter with subclinical mastitis Holstein dairy cows were selected from several cattle farms in Isfahan. which divided into three groups: positive control, negative control, and those treated with the extract.

Sample Collection

The selection process for cows in the study involved examining their monthly records and testing the milk of selected cows (those with a calving abdomen count between 2 and 4 and somatic cell count above 400,000) using the California Mastitis Test (CMT). For the CMT, 2 ml of milk from each quarter was collected separately and placed in the wells of the CMT plate. Then, 2 ml of CMT solution was added to each well as per the manufacturer's instructions, ensuring thorough mixing of the solution and milk. The results were read within 20 seconds. Milk samples with CMT scores of trace, 1, 2, or 3 (indicating positive reactions) were aseptically collected before milking.

After confirming the presence of subclinical mastitis and ensuring the absence of symptoms associated with acute and clinical mastitis (such as fever, loss of appetite, pain, stiffness, redness of the udder, or changes in milk appearance), the udder teats were washed with warm water, disinfected with a Predip solution, and dried using a paper towel after 30 seconds. The teats were then wiped with a napkin soaked in 70% alcohol for 30 seconds. Next, three initial milkings were conducted, and 10 ml of milk was collected in a sterile sample container for bacteriological culture and somatic cell count. It is important to note that the milk sampling container was positioned at a 45-degree angle to the udder to prevent the entry of external factors such as dust into the container. Contaminated milk samples were cultured using BHI Broth medium, and biochemical and molecular PCR tests were conducted to isolate and accurately identify the bacteria present. The analysis revealed the presence of Escherichia coli and Staphylococcus aureus bacteria in the milk samples.

Extraction:

For the extraction of the mangrove plant (Avicennia marina), mature leaves were collected from Khamer port in Hormozgan, which is a natural habitat for mangroves. The leaves were green and healthy, and they were washed with distilled water before being dried in the shade for one week. Once dried, the leaves were ground into a powder using an electric mill. To prepare the extract, 100 grams of the leaf powder was mixed with 400 cc of 96% ethanol in a container and placed on a shaker for 72 hours. After the designated time, the mixture was separated using Whatman filter paper, and the resulting supernatant was transferred to a rotary device to remove the solvent (ethanol) and concentrate the extract at 45°C. The concentrated solution was then poured onto sterile petri dishes and allowed to dry in the air. UV rays were used to irradiate the dried extract, and it was stored in a refrigerator.

As for the spirulina algae, sampling was conducted at tidal stations located on rocky and sandy beaches in Ramin, Chabahar. The collected algae samples were stored in nylon bags containing a small amount of seawater and transported to the laboratory. In the lab, the algae samples were thoroughly cleaned and washed with double-distilled water. The algae were immersed in distilled water twice, with the water being changed every three hours over a 24-hour period to remove excess salts from the tissues. Afterward, the algae samples were dried in an oven at 60°C for 24 hours. Once dried, the samples were ground into a powder using an electric mill. The extraction process was carried out using the ethanol method and a 10% mass-volume immersion technique. For this, 40 grams of powdered algae samples were weighed and transferred to glass containers. Then, 200 ml of ethanol was added to the containers to prepare the ethanolic extract. The containers were shaken and kept in a dark environment for 48 hours. Every 16 hours, the solution was stirred for 25 minutes to facilitate better and more effective extraction of the desired substances within the algae tissues. After the designated time, the solutions were filtered using Whatman No. 1 paper. The filtered liquid was desolvated using a rotary evaporator under vacuum conditions at a temperature of 50°C. The concentrated extracts were then sterilized using a 0.22-micron microbial filter. Prior to the extraction experiments, all containers and glassware were carefully washed and labeled.

Preparation of injectable ointment:

Dried extracts of mangrove plant and Spirulina algae were applied on Petri dishes at doses of 300mg (6MIC) and 90mg (6MIC) respectively. Under sterile conditions, the extracts were scraped from the bottom of the Petri dishes and then mixed with 10 ml of liquid paraffin as an ointment base using a magnetic stirrer. To ensure lubrication, 5 cc of distilled water was added to the mixture.

The pharmaceutical form of the medication is an intramammary ointment. The drug composition of a 10 ml syringe includes 300 mg of ethanolic extract of Spirulina and 90 mg of ethanolic extract of mangrove, along with 10 ml of liquid paraffin.

The medicine is used for the prevention of subclinical mastitis and the prevention of new bacterial infections in the udder caused by organisms sensitive (Staphylococcus aureus and Escherichia coli) to this medication. The entire contents of the syringe, containing 300 mg of ethanolic extract of Spirulina, 90 mg of ethanolic extract of mangrove, and 10 ml of liquid paraffin, are to be used for each quarter and in each treatment

Advantage effects of invention

Effectiveness: The effectiveness of the invention has been demonstrated through laboratory tests and statistical analysis conducted in cattle breeding. The results provide evidence of its efficacy in treating subclinical mastitis.

Elimination of Antibiotics: The invention eliminates the need for antibiotics to treat subclinical mastitis in cattle farms. This is a significant advantage as it helps reduce the reliance on antibiotics, addressing concerns such as antibiotic resistance and potential residues in milk and dairy products.

Cost-Effectiveness: The invention is economical and reduces treatment costs associated with subclinical mastitis. This can have a positive impact on the financial aspects of cattle farming and improve overall profitability.

Avoidance of Other Antiseptics: Unlike other treatments that may utilize antiseptics like formalin, the claimed invention avoids the use of such substances and the problems associated with them. This ensures a safer and more desirable treatment option.

Ease of Use: The invention is designed for ease of use and consumption by dairy workers. It does not require specialized expertise, making it accessible to a wider range of individuals involved in cattle farming.

Constant Availability: The invention offers the advantage of constant availability in the domestic market. This eliminates the need to rely on imported mastitis treatment drugs, saving currency and ensuring a steady supply of the necessary treatment.

Absence of Antibiotic Residues: By not using antibiotics, the invention ensures the absence of antibiotic residues in the produced milk and other dairy products. This promotes the healthiness and safety of these products for consumers.

: Chemical structure of important compounds in mangrove plant

: Chemical structure of phycocyanin in spirulina algae

: flowchart of product

: Chemical structure of important compounds in mangrove plant

: Chemical structure of phycocyanin in spirulina algae

: flowchart of product

Examples

Determining the antibacterial activity of the extracts:

To assess the antibacterial activity of the ethanolic extract of mangrove leaves and the ethanolic extract of spirulina algae, the well plate test and microdilution method were utilized. These methods aimed to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extract

Determining the minimum concentration of ethanol extract of mangrove leaves

MBC(mg/ml)	MIC(mg/ml)	Bacteria
150	50	Staphylococcus aureus
32	8	Escherichia coli

Determination of minimum ethanolic extract of spirulina algae

MBC(mg/ml)	MIC(mg/ml)	Bacteria
30	15	Staphylococcus aureus
14	6	Escherichia coli

Agar Well diffusion of ethanolic extract of Avicenna marina

The antibacterial property of the ethanolic extract of Avicennia marina (mangrove plant) was evaluated using the Agar Well diffusion method. To begin, a suspension equivalent to half of the McFarland standard (1.5 * 108) was prepared from Staphylococcus aureus bacteria in a tube containing physiological serum. The bacterial suspension was then evenly spread over the surface of Mueller Hinton Agar plates (Merck-Germany) using a sterile swab.

Next, sterile punches were made on the agar surface, and 100 microliters of the mangrove plant extract at concentrations of 43, 87, 175, 350, and 21 mg/ml were introduced into the wells. The plates were then incubated at 37°C for 24 hours to allow for bacterial growth and the diffusion of the extract.

After the incubation period, the diameter of the inhibition zones around the wells, which indicated the extent of bacterial growth inhibition, was measured and recorded using a specialized ruler. This measurement provided an indication of the antibacterial effectiveness of the ethanolic extract of Avicennia marina against Staphylococcus aureus.

Agar Well diffusion of ethanolic extract of avicennia marina

Concentration of mangrove plant extract (mg/ml)	300	175	87	43	21
Zone of inhibition in mm	13	10	0	0	0	s.aureus
Zone of inhibition in mm	24	18	5/13	5/3	0	e.coli

Agar Well diffusion of ethanolic extract of Spirulina platensis algae:

In this method, the antibacterial activity of the extract was assessed using the Agar Well diffusion technique. After preparing the microbial suspension, a lawn culture of bacteria was spread evenly on the surface of Mueller Hinton agar, which served as the culture medium. Wells with a diameter of 6 mm were then created on the agar surface using the end of a sterile Pasteur pipette. These wells were spaced at least 2.5 cm apart and kept away from the edges of the plate.

Next, 50 microliters of the extract, at concentrations of 2.5 and 5 mg/ml, were carefully added to the respective wells. The plates were then transferred to an incubator and maintained at 37°C for 24 hours to allow for bacterial growth and the diffusion of the extract into the surrounding agar medium.

After the incubation period, the diameter of the clear zones around the wells, where bacterial growth was inhibited, was measured using a ruler. This measurement provided information on the sensitivity or resistance of the bacteria to the extract's components. Larger diameter halos indicated greater antibacterial activity and increased bacterial sensitivity to the extract.

Agar Well diffusion of ethanolic extract of Spirulina platensis algae

Concentration of ethanol extract of spirulina algae (mg/ml)	5	5/2	type of bacteria
Zone of inhibition in mm	13±0.4	9±0.5	s.aureus
Zone of inhibition in mm	16±0.6	14±0.5	e.coli

To establish the efficacy of this compound in controlling mastitis in an INVIVO environment, the compound was directly injected into the udder of a dairy cow. This study involved 75 quarter cows with subclinical mastitis from Holstein dairy farms in Isfahan and divided into three groups: positive control, negative control, and those treated with the extract. The selection of cows for the study involved checking their monthly records, specifically focusing on cows with 2 to 4 calvings and somatic cell counts over 400,000. Additionally, the presence or absence of clinical signs of mastitis was assessed by the farm veterinarian.

Implementation Method:

The herbal ointment, formulated as a result of the invention, can be used to treat subclinical mastitis. The recommended method is to apply the ointment intramammary once a day for three consecutive days. Antibiotics are not required in this treatment approach.

The method involves intramammary injection, with the infected areas being treated three times, with a 24-hour interval between each treatment. It is important to completely drain the breast milk before injection and ensure thorough disinfection and cleaning of the pacifier heads. Care should be taken to prevent contamination of the injection site. Each syringe should only be used once to maintain hygiene and effectiveness.

Time to avoid consumption

Meat: 0 days Milk: 0 days

Industrial Production Method:

To prepare the extract from mangrove leaves, 100 grams of mangrove leaf powder is mixed with 400 cc of 96% ethanol. The mixture is then placed on a shaker for 72 hours. Afterward, the supernatant is separated using Whatman filter paper, and the solvent is removed from the supernatant using a rotary device at a temperature of 45°C. The resulting solution is poured onto sterile Petri dishes and dried in the air. Finally, the dried extract is exposed to UV rays and stored in a refrigerator.

For the extraction process of the spirulina algae, 40 grams of powdered algae samples are weighed and placed in glass containers. 200 ml of ethanol is added to the containers, and the mixture is shaken and kept in a dark environment for 48 hours. The solution is stirred every 16 hours for 25 minutes to enhance the extraction process. The solution is then filtered using Whatman No. 1 paper, and the filtered liquid is desolvated using a rotary evaporator under vacuum at a temperature of 50°C. The concentrated extracts are sterilized using a 0.22-micron microbial filter.

The herbal ointment is prepared by combining the alcoholic extract of mangrove medicinal plant at a ratio of 300mg and the alcoholic extract of spirulina algae at a ratio of 90mg per liter. This extract is mixed with a paraffin carrier. The ointment can be packaged in tubes and is administered as an intramammary injection for the treatment of subclinical mastitis in cattle.

[Table. 5] Fatty acid content and lipid composition of Spirulina

[Table. 6] Amount of fatty acids composition of spirulina lipid complex

[Table. 7]: Antimicrobial mechanism of biochemical compounds of spirulina algae

Table 3: Antimicrobial mechanism of biochemical compounds of spirulina algae

[Chart. 1]: The composition of the bacterial population before and after the injection of compound 1

[Chart. 2]: Measurement of somatic cells before and after injection of compound 1

Chart 3: The composition of the bacterial population before and after the injection of compound 2

[Chart. 4]: Measurement of somatic cells before and after injection of compound 2

[Chart. 5] and [Chart. 6]: The composition of the bacterial population and the measurement of somatic cells before and after the injection of compound 3

[Chart. 7]: Bacterial population composition before and after normal saline injection

[Chart. 8]: Measurement of somatic cells before and after using normal saline (negative control group)

[Chart. 9]: Bacterial population composition before and after Cefquinum antibiotic injection (positive control group)

[Chart. 10]: Measurement of somatic cells before and after the use of Cefquinum antibiotic

The invention has significant industrial applications in the field of pharmaceuticals and veterinary medicine, specifically for the treatment of subclinical mastitis in cattle. It can be utilized by pharmaceutical companies and veterinary clinics to produce effective treatments for this condition.

Claims

A process for preparing an injectable herbal ointment for the treatment of subclinical mastitis, comprising:
Combining 300 mg of ethanolic extract of spirulina algae (Platensis Spirulina) and 90 mg of ethanolic extract of mangrove leaves (Avicennia marina) in each 10 ml tube of the ointment.
Adding 10 ml of sanitary liquid paraffin as a carrier.
The process according to claim 1, wherein the mangrove plant extract is prepared by the following steps:
Soaking 100 grams of mangrove leaf powder in 400 cc of 96% ethanol.
Placing the mixture on a shaker for 72 hours.
Separating the supernatant using Whatman filter paper.
Concentrating the supernatant to a thick crude extract using a rotary vacuum evaporator at 45°C.
Pouring the solution onto sterile Petri dishes and drying it in the air.
Exposing the dried extract to UV rays and storing it in the refrigerator.
The process according to claim 1, further comprising the preparation of the Spirulina platensis algae extract by the following steps:
Soaking 40 grams of algal powder in 200 cc of 96% ethanol.
Stirring the desired solution every 16 hours for 25 minutes.
Filtering the prepared solutions with Whatman No. 1 paper.
Concentrating the filtered liquid using a rotary vacuum evaporator at 50°C.
Sterilizing the extracts with a 0.22-micron microbial filter.
The process according to claim 3, wherein the bottles containing the solvent and algal powder are shaken during extraction and kept in a dark environment for 48 hours.
The process according to claim 3, wherein the solvent used in the extraction process is ethanol.
The process according to claim 1 and claim 2, wherein the extraction process is carried out using the ethanol method with a 10% mass-volume immersion technique.
The process according to claim 2, wherein the solvent extraction is performed using a rotary vacuum evaporator at 45°C.
The process according to claim 3, wherein the solvent extraction is performed using a rotary vacuum evaporator at 50°C.
The process according to claim 1, wherein the extracts are sterilized using a 0.22-micron microbial filter.
The ointment according to claim 1 is supplied as an intramammary ointment for the treatment of subclinical mastitis.