OA20768A - Improved traditional medicine antibacterial - antiviral and manufacturing method. - Google Patents

Abstract

The product in the form of an oral solution and in the form of capsules is a new composition based on decoction extract and crude extract of Jairopha gossypifolia. This improved traditional medicine (MTA) is used in the treatment of bacterial and viral diseases, including digestive and urinary tract infections. It acts on a wide range of bacteria consisting of Escherichia coli ATCC 35218, Pseudomonas aeruginosa, Staphylococcus aureus ATCC 25923, Shigella sonnei, Salmonella typhi, Proteus vulgaris, Kleibsiella pneumonia, Klebsiella oxytacoe, Aeromonas hydrophila, Vibrio cholerea, Citrobacter freundii, Citrobacter diver sus , Enterobacter aerogenes. Based on chemical analyses, its toxicity and safety, the predominant secondary metabolites in the extracts consist of tannins, flavonoids, terpenoids and steroids. Good activity resulting in MICs of between 39 and 1250 was demonstrated on the 13 bacterial species. Furthermore, no acute or subacute toxicity was observed with the tolerance of the extracts at high lethal doses (LD) greater than 5 g/kg of body weight of the experimental mice. The dose of 2 to 5 g/l of the plant extract is suitable for formulating a traditionally improved antibacterial and antiviral drug in the form of an oral solution and in the form of capsules.

Description

DESCRIPTION OF THE INVENTION

1. Field of the invention

The present invention relates to the formulation and packaging of an improved traditional medicine (MTA) from the leaf extracts of Jatropha gossypifolia. The antimicrobial and antiviral 5 MTA is administered in the form of an oral solution and in the form of capsules, in particular for the treatment of pulmonary and digestive tract infections (Cough, Covid-19, bronchitis, pneumonia, tuberculosis, cholera, typhoid fever, viral hepatitis B & C), infections caused by diabetes-related complications, skin rashes such as shingles, HIV-AIDS-related opportunistic illnesses (10 infections caused by a lowered immune system) contributing to a dramatic drop in viral load .

2. Prior Art

Antibiotic resistance today has become a worldwide concern, so that the clinical efficacy of many existing antibiotics is threatened by the emergence of multi-resistant strains of bacteria. The use of medicinal plants with the use of extracts made up of a set of chemical compounds, which will act in synergy, constitutes an alternative of choice to deal with this problem of multi-resistance. There is a continuous and urgent need to discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action for these infectious diseases.

Indeed, many infectious diseases have been known to be treated with medicinal plants in different traditional African communities. This is the case of Jatropha gossypifolia L, with regard to its multiple traditional uses, which is a plant whose antimicrobial and antiviral activity has been demonstrated.

It is a small bushy shrub up to 4 m high, with very decorative foliage, purple and yellow inflorescences, of the Euphorbiaceae family. It contains a pinkish milky substance called latex. The leaves consist of a petiole 8 to 12 cm long, furnished with glandular hairs. The stipules about 5 mm long are found at the base of the petiole divided into several segments.

Throughout tropical Africa different parts of J. gossypifolia are used for a variety of medicinal purposes. In Senegal a leaf decoction is taken against colic, stomach-ache and fever. In Ghana the leaves are used as a purgative, and leaf sap is applied to the tongues of babies to treat thrush, and of adults against inflammation. The marrow of old stems is put in the nostrils to induce sneezing and thus cure headaches.

Phytochemical investigations revealed the presence of lignans (e.g. gadain, 5-jatrodiene, gossypifane, gossypidiene and prasanthalin) in the light oil extract of stem, root and seeds; the alkaloid jatrophine in the juice, and flavonoids (apigenin, vitexin and isovitexin), as well as triterpenes in the ethanoic leaf extract. With regard to antimicrobial activity, the antibiotic activity of the various extracts of J. gossypiifolia is frequently reported. the related species Jatropha curcas. Very few data are available on the antiparasitic efficacy of J. gossypiifolia like macrocyclic jatrophenone, a diterpene, which has significant antibacterial activity in vitro against Staphylococcus aureus. Furthermore, the efficacy of extracts from this plant n has not yet been tested on a wide range of bacterial strains for the formulation of a phytomedicine

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3. protocol of the invention

3.1 Preparation of plant material

The leaves of Jatropha gossypiifolia were harvested from mature plants, approximately 1.7 to 20.2 m high, having fructified in a crop field in the city of Yaoundé at the place called the barrier. Once cut, the leaves were dried in the shade, at room temperature, for 6 days, then crushed to obtain a powder.

3.2, Extraction process and phytochemical screening

Leaf powder due to 500 g was introduced into 5 1 of water. The contents were boiled for 25 min; then left to cool for 1 h. The decoction extract was filtered under hydrophilic cotton then concentrated. In order to determine the concentration of the extract solution, 0.5 1 of the decoction was dried in an oven at 40°C for 2 days. The mass obtained (2.5 g) after drying gave a final concentration of 5 g/L of the decoction extract.

The phytochemical screening was carried out on the concentrated extract based on the characteristic tests of certain functional groups described by Harbone (1998)'. The results revealed the presence of saponins, tannins, flavonoids, reducing sugars, ¹ Harbone JB 1998. Textbook phytochemical methods. A guide to modem techniques of plant analysis. 5th

Edition, Chapmanand Hait. Td. London. 21-72.

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I I cardiac glycosides, terpenoids, triterpenoids, steroids, macrocyclic Jatrophenone diterpene a compound very active on bacteria and viruses, proteins and starch. On the other hand, alkaloids and coumarins were absent in the leaves. The chemical constituents present are known to have pharmacological activities, particularly on various bacterial species and strains.

3.3. Study of antibacterial activity

3.3.1. Preparation of the extract solution

The crude extract was collected using a 10 ml syringe and filtered through a whatmann membrane with a porosity of 0.45 micrometers. Then, 100 µl of the filtered extract sample was cultured on each specific medium. The reference antibiotic used was gentamicin.

3.3.2. Preparation of Microbial Finoculum

For activation, a few bacterial colonies (2 to 4) were removed from the preservation medium [Plate Count Agar (PCA)] using a sterilized loop and spread on the surface of Miller Hilton Agar (MHA) medium. previously poured into 30 mm Petri dishes after 18 h incubation at 37°C. The bacterial suspensions were prepared by taking colonies from this culture, which were diluted in sterile distilled water containing 0.9% NaCl until a turbidity corresponding to point 0.5 on the scale was obtained. of Mc Farland (Table 1) corresponding to the concentration of 1.5x10 ⁸ CFU/mL (Kuete et al., 2010).

3.3.3. Microorganisms

Thirteen bacterial species were used, namely:

- Gram ⁺ bacteria _: Staphylococcus aureus ATCC 25923;

- Gram' bacteria: Escherichia coli ATCC 35218, Pseudomonas aeruginosa, Shigella sonnet, Salmonella typhi, Proteus vulgaris, Kleibsiella pneumonia, Klebsiella oxytacoe, Aeromonas 25 hydrophila, Vibrio cholerea, Citrobacter freundii, Cilrobacter diversus, Enterobacter aerogenes

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3.3.4. Evaluation of antibacterial activity

The activity was evaluated on the total extract of the water decoction of J. gossypifolia, using the methods of diffusion and dilution in agar, following the technique of the National Committee for Clinical Laboratory Standards ⁽²³⁾ .

Determination of the minimum inhibitory concentration (MIC) in liquid medium: The microplate method was described by (Newton et al. (2002). A volume of 100 pL of Mueller Hinton broth was introduced into all 96 wells of the microplate as indicated by (Jennifer M. Andrew. ¢2001), Newton et al., 2002, Naïtali & Dubois Brissonnet 2017). Then in the first wells of each column was introduced a volume of 100 μΐ of each stock solution of extract at a concentration of 5000 mg/mL. Successive serial dilutions of ratio 2 made it possible to obtain a range of concentrations going from 5000 mg/mL to 19 mg/mL. From a young culture of 24 hours on agar medium poured on a slope, a microbial suspension of each germ was prepared in sterile physiological water (NaCl, 8.5%) by adjusting the turbidity to that of the solution of 0.5 MacFarland (Add 0.5 mL of

0.0048M Bacl2 (1.17%w/v Bacl2. 2H2O) to 99.5mL of 0.18M H2SO4 (1% v/v) i.e. 1.5* 10 ⁸

CFU/mL for bacteria and 2 ^χ 10 ⁸ CFU/mL for yeasts (Hang M. (1999), Jennifer M. Andrew. (2001), Kuete et al., 2010). A first dilution was then made to obtain intermediate solutions at 10 ⁷ CFU/mL and 10 ⁶ CFU/mL respectively. Then a second dilution to obtain inocula at 10 ⁶ CFU/mL and 10 ⁵ CFU/mL. 100 pl of each inoculum was subsequently introduced into each well, for a final volume of 200 μΐ per well. The positive control was placed in columns 11 and 12. This positive control consisting of Gentamicin or iluconazole, was prepared under the same conditions as the extract and tested at a concentration of 08 successive dilutions between 5000 to 39 mg /mL (Houssou Raymond Fatondji et al., 2010). The negative control consisted of a mixture (culture medium+bacterial inoculum in the proportions (1/1; V/V)). The wells containing only the culture medium were reserved for the sterility control. The microplates were incubated at 37°C for 18 to 24 hours. After incubation, bacterial growth was revealed using iodonitrotetrazolium chloride (INT), the principle of which is based on the capture of protons emitted by dehydrogenase enzymes present on the

National Committee for Clinical Laboratory Standards. (1997a). Performance standards for antimicrobial disk susceptibility tests. ^6'h ed. Approved standard M, A _s (M _]œ -S ₇ ). NCCLS, Wayne, Pa.

³ National Committee for Clinical Laboratory Standards. (1997b). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4 ^Λ ed. Approved standard M ₂ A ₄ (M _lû0 -S ₇ ).

NCCLS, Wayne, Pa.

membrane of live bacteria (Houssou Raymond Fatondji et al., 2018). 40 μΐ of the INT solution were thus introduced into each of the wells of the microplate. After glucose metabolism, a change in the medium to pink-red after 30 min of reincubation reveals an insufficient concentration of the metabolite to inhibit bacterial growth. In the unstained well and containing the lowest amount of secondary metabolite, the concentration of the extract corresponds to the MIC (Matîvandleda et al., 2006, Houssou Raymond Fatondji et al., 2018). The MIC corresponds to the concentration of the well not colored in red and in which there is the lowest quantity of secondary metabolite. The tests were repeated in triplicate.

Determination of the minimum bactericidal concentration (MBC) in liquid medium: The 150 µL volume of sterile medium (Mueller Hinton Broth) was introduced into the wells of another microplate, also sterile and different from that of the CMIs. Then we added 50μ1 of extract taken from the wells corresponding to the MICs obtained. We also took 50 pL of extract from the wells that preceded those with the MIC (ISO standard 20766-1: 2006). The sterility control consisted of 200 μl of medium (Mueller Hinton Broth). The positive control consisted of Gentamicin, treated like the extracts. The negative control was medium without extract. The CMB was represented by the well corresponding to the lowest concentration of extract which showed no visible bacterial growth (EC 1997).

CMB/CMI ratios: The CMB/CMI ratios made it possible to confirm the bacteriostatic or bactericidal nature of the extracts tested. When this ratio is greater than 4, the substance is said to be bacteriostatic. If this ratio is less than 4, the substance is considered bactericidal (Oussou et al., 2008; Abedini, 2013).

Determination of the sensitivity of germs to the extracts tested: The method of diffusion in agar medium from disks was used (Mathabe et al., 2006; Shan et al., 2007). Principle: Using forceps, whatmann paper discs 7 mm in diameter are placed in Mueller Hinton agar poured into Petri dishes. Inoculations with the bacterial suspensions are then carried out; on each disc 50 μΐ of extract was deposited on the disc. After 24 h of incubation at 37°C in an oven, the diameters of the zones of inhibition are measured. The extract was tested at a concentration of 5000 mg/ml. The tests were replicated three times.

Results of the antibacterial activity test: i) Diffusion in agar: Bacteria, whether gram+ or gram-, were generally sensitive to their treatment with plant extracts (Figure 1).

Figure 1. Inhibition diameter of extracts against some bacterial strains

a) Aeromonas hydrophila, b) Shigella sonnei; c) Klebsiella oxytacoe

Table 1 shows that the inhibition diameters (ID) are between 16 and 25 mm. Overall, the bacterial strains are sensitive in the presence of the extracts. No weak activity (DI was recorded. On the other hand, only one average activity (8 < DI < 15) was recorded with Citrobacter freundii whereas the activity of the extracts was strong (DI ^5) on the twelve others bacterial species In addition, the ID of the extract on the majority of the bacterial species is higher than that of the control.

ii) Minimum Inhibitory Concentrations (MIC) and Bactericidal Concentrations (CMD); The MICs of the extracts are between 39 (Salmonella typhi) and 1250 mg/L (Klebsiella pneumonia and Shigella sonnei) while the CM B are between 39 (in Staphylococcus aureus) and 156 mg/L. By comparing the CMB with the MIC of the active extracts according to the 20 scale obtained by Gatsîng and Adoga (2007) ⁴ on the Cassia petersiana plant when CMB/CMI >4 there is a bacteriostatic effect, CMB/CMI < 4 the effect is bactericidal. Thus the extracts have a bactericidal effect on 10 bacterial species against only two bacteriostatic effects for 2 species of bacteria (Salmonella typhi and Vibrio cholered) (Table 1).

⁴ Gatsing D, Adoga G 1. (2007). Antisalmonella activity and phytochemical screening of various parts of Cassia petersiana (Cesulpiniaceae). Research Journal of Microbiology, 2:876-880

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ΤΟ: Negative control (physiological water); Tl: Positive control (Gent: Gentamicin)

3.4 Acute toxicities

All of the effects were tested, on animals used as experimental models, by short-term exposure (14 days) to a high dose (concentration) of J. gossypiifolia extract. The oral lethal dose (LD50) was determined according to the guidelines for oral toxicity studies: 423 of the Organization for Economic Cooperation and Development (OECD). Male and female Swiss albino mice (25-30g) and Wistar rats (170-210g) bred in the animal facility of the Institute for Medical Research and the Study of Medicinal Plants (IMPM) were used for the evaluation. acute toxicity.

Five doses of the plant extract from 1000 mg/kg to 10000 mg/kg were administered to the mice. They were followed for 14 days to track their moaning, sniffling, breathing, convulsing, abdominal gait and their responsiveness to the environment. The lethal dose (LD50) was then determined. The results showed that the extract is well tolerated at high doses with lethal doses 50 greater than 5000 mg/kg. No mortality was recorded even at the maximum dose of 5000 mg/kg per body weight for the extract. Moreover, the tested animals showed no significant behavioral change compared to the control at the end of the 14 days of general observation. It can be stated that the decoction of J. gossypiifolia is not toxic orally in acute administration.

| 20 3.5. Pharmaceutical form

3.5.1 Good practices | The Battioli manual consisting of preparation standards, magisterial, officinal, phytotherapeutic and homeopathic formulations were used as the basis for the | galenic formulation.

3.5.2 Preservatives | Sodium benzoate and the essential oil of Cilrus sinensis, Syzygium aromaticum,

Nigella sativa, Citrus Sinensis peel, Manihot esculenta starch were used | as a preservative, taking care to check their physico-chemical parameters (weight, expiry date, storage conditions, etc.). The effective dose was determined based on the results of toxicity and antimicrobial activity.

I 3.5.3 Protocol for manufacturing the oral solution

The oral solution was prepared from the filtrate of the decoction of the aqueous extract of Jatropha gossypiifolia contained in polystyrene bottles of five hundred milliliters (500

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1 ml) of final product. No incompatibility was observed between the filtrate of the extract | aqueous; clove, black cumin, mixed with sodium benzoate and orange essential oil which go into the preparation of the oral solution.

| The mixture in permanent homogenization was carried out in the following order: filtrate of the decoction of the aqueous extract, clove, black cumin, orange essence and sodium benzoate.

Five hundred milliliters (500 ml) of oral solution were distributed per polystyrene bottle. The labels were then stuck on said bottles.

Table 2 shows the composition of the extract solution per 500 mL.

Table 2. Composition of the 500 mL oral solution

Plant extract of Jatropha gossypifolia 2.5g sodium benzoate 0.1g Distilled water 500mL Syzygium aromaticum 0.5g Nigella sativa 0.3g Essence of Citrus sinensis 0.2g

NB: this method of manufacturing the oral solution is reproducible for the capacities of 1000ml, 2000ml and 5000ml.

3.5.4 Capsule manufacturing protocol

The capsules were prepared from the crude extract of Jatropha gossypifolia contained in 20 capsules of 500mg per unit either in total or in total 60,000 mg for 120 capsules and 30,000 mg for 60 capsules. No incompatibilities were observed between the crude extract; mixed with cloves, black cumin, orange zest and cassava starch which go into making the capsules.

The mixture in permanent homogenization was carried out in the following order: Raw extract, clove, black cumin, orange zest and cassava starch.

000 mg of the mixture were distributed in capsules of 500 mg for a bottle of 120 capsules and 30,000 mg for bottles of 60 capsules.

Table 3 shows the composition of the mixture for a 500 mg capsule.

Table 3. Composition of the 500 mg capsule

Raw plant extract of Jatropha gossypifolia 350mg Syzygium aromaticum 50mg Nigella sativa 40mg Zest of Citrus sinensis 30mg Manihot esculenta starch 30mg

NB: This process is reproducible for as many capsules to be manufactured and according to the capacities of the bottles.

3.6, Dosages

3.6.1 Adult dosage a- Oral solution

It is recommended to consume 02 to 03 tablespoons of product or even % glass of product depending on the case for adults.

b- Capsules

The adult dosage is 02 capsules morning and evening for adults.

3.6.2 Child dosage a- Oral solution

It is recommended to consume 01-02 tablespoons of the product for children.

b- Capsules

The child dosage is 01 capsule morning and evening.

Claims (10)

1. Improved traditional medicine, effective against bacterial and viral infections, characterized in that it is prepared from the aqueous extract and the crude extract of Jatropha gossypifolia. 2. Improved traditional medicinal product, according to claim 1, characterized in that it contains for the preparation of 5000 mL of aqueous solution: 500 g of Jatropha gossypifolia leaf powder and for a 500 mg capsule: 350 mg of Jatropha gossypifolia powder. Jatropha gossypifolia leaf, which is equivalent to 60,000 mg for 120 capsules, or 42,000 mg of Jatropha gossypifolia powder; 30,000 mg for 60 capsules, i.e. 21,000 mg of Jatropha gossypifolia powder. 3. Improved traditional medicine according to claims 1 and 2 is characterized in that it contains mostly saponins, tannins, flavonoids, terpenoids, triterpenoids, steroids and the major active compound isolated macrocyclic diterpene Jatrophenone with significant activity against Gram ⁺ bacteria _: Staphylococcus aureus ATCC 25923 and Gram* bacteria: Escherichia coli ATCC 35218, Pseudomonas aeruginosa, Shigella sonnei, Salmonella typhi, Proteus vulgaris, Kleihsiella pneumonia, Klebsiella oxylacoe, Aeromonas hydrophila, Vibrio cholerea, Citrobacter freundii , Citrobacter diversus, Enterobacter aerogenes. 4. Improved traditional medicinal product according to claims 1,2 and 3 characterized in that it is presented in the form of a drinkable solution of 500 ml & 1000 ml and bottles of 60 & 120 capsules. 5. Oral solution of 500 ml according to claim 4 characterized in that it is constituted as follows: - Plant extract from the decoction of Jatropha gossypiifolia ·. 2.5g - Sodium Benzoate: 0.1g - Distilled water: 500 mL - Syzygium aromaticum: 0.5g -Nigella sativa: 0.3g - Essence of Citrus sinensis: 0.2g 6. Bottle of 60 and 120 capsules according to claim 4 characterized in that it is made up as follows for each 500 mg capsule: - Crude extract of Jatropha gossypifolia: 350 mg - Syzygium aromaticum: 50 mg - Nigella saliva: 40mg - Citrus sinensis: 30mg - Manihot esculenta starch: 30 mg 7. Improved traditional medicinal product according to any one of claims 5 and 6, characterized in that the drinkable solution and the galenic presentation in the form of capsules are reproducible according to the capacities to be obtained, i.e. 1000 ml, 2000 ml, 2500 ml and 5000 ml and according to the number of capsules to be produced and the capacity of the bottles. 8. Improved traditional medicinal product according to any one of claims 1, 5 and 6, characterized in that the drinkable solution and the galenic presentation in the form of capsules is indicated as an antibacterial and antiviral, in particular against pulmonary and digestive tract infections (cough , covid-19, bronchitis, pneumonia, tuberculosis, cholera, typhoid, viral hepatitis B & C), infections caused by complications related to type 2 diabetes, skin rashes such as Shingles opportunistic diseases related to HIV-AIDS ( infections caused by the weakening of the immune system) contributing to the considerable drop in viral load. 9. Improved traditional medicinal product characterized in that the appropriate dosage for the oral solution is: 02 to 03 tablespoons or even 'Λ (half) or 1 (one) glass of product depending on the case of the disease morning and evening for adults; 01 (one) to 02 - (two) tablespoons for children and this dosage is in both cases half as a preventive cure depending on the disease. 10, Improved traditional medicine characterized in that the dosage adapted for the capsules: 02 capsules morning and evening for adults; 01 capsule morning and evening for children and this dosage is in both cases half as a preventive cure depending on the disease.