RHOEAS RED PETALS

TECHNICAL FIELD

This invention is a natural antiviral and anti-inflammatory compound that effects on SARS CoV-2 (Covid-19) and Influenza virus (H1N1, H5N1) consist of specific bioflavonoids which extracted from Papaver rhoeas red petals.

PRIOR ART

Flavonoids, including chalcones, flavones, flavonols, anthocyanins and proanthocyanidins, are widely distributed in the plant kingdom and their metabolic pathways have been extensively studied using both biochemical and molecular techniques .

Flavonoid compounds derived from plants remain an important resource for the discovery and development of new antiviral drugs due to their expected low side effects and high availability in nature.

Antiviral activities of various flavonoids have been reported against some viruses including coronavirus, influenza virus, human cytomegalovirus (HCMV), herpes simplex virus 1-2, dengue virus, human adenoviruses etc.

Coronaviruses (CoVs) are an etiologic agent of severe infections in both humans and animals, which can cause disorder not only in the respiratory tract but also in the digestive tract and systemically. The new strain of CoV was identified at the end of 2019, initially named 2019- nCoV, and emerged during an outbreak in Wuhan, China. WHO named this CoV infections as COVID-19 (coronavirus disease 2019), on February 11, 2020. Currently, no specific therapies for COVID-19 treatment. However, the limited supportive therapies are designed to prevent further complications and organ damage. Some preliminary studies have investigated potential combinations that include the protease inhibitor lopinavir/ritonavir, which is commonly used to treat human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome patients, for the treatment of COVID-19-infected patients. Other reported antiviral treatments form human pathogenic CoVs include nucleoside analogues, neuraminidase inhibitors, remdesivir, umifenovir (arbidol), tenofovir disoproxil (TDF), and lamivudine (3TC). Also nelfmavir was identified as the best potential inhibitor against COVID-19 M protease. Siti Khaerunnisa et all investigated some flavonoids (includes kaempferol, quercetin, luteolin-7-glucoside, demethoxycurcumin, naringenin, apigenin-7-glucoside, oleuropein, curcumin, catechin, epicatechin-gallate, zingerol, gingerol, and allicin) as potential inhibitor candidates for COVID-19 Mpro. They suggested that nelfmavir and lopinavir may represent potential treatment options, and kaempferol, quercetin, luteolin-7- glucoside, demethoxycurcumin, naringenin, apigenin-7-glucoside, oleuropein, curcumin, catechin, and epicatechin-gallate were the most recommended flavonoid compounds that may act as potential inhibitors of COVID-19 Mpro.

Flavonoids have also been shown in a number of studies to be potent antioxidants, capable of scavenging hydroxyl radicals, superoxide anions, and lipid peroxyl radicals. Since the most important aspect in viral disease treatment is to inhibit virus replication, flavonoids as antiviral and antioxidant activities should be treatment of severe influenza-associated complications. Uchide and Toyoda used flavonoids compounds, such as 5,7,4-trihydroxy-8- methoxyflavone, catechins, quercetin 3-rhamnoside, isoquercetin and oligonol, possess both antiviral and antioxidant activities. In their theory, combination of these antioxidants with current anti-influenza drugs could improve conventional chemotherapy for severe influenza- associated complications. As Yao Li et all reported that, Quercetin is a long lasting anti-inflammatory substance. This strong anti-inflammatory potential can be expressed on different cell types, both in animal and human models. It is known that Quercetin has both mast cell stabilizing and gastrointestinal cytoprotective activity. It can also play a modulating, biphasic and regulatory action on inflammation and immunity. Papaver rhoeas is an annual herb indigenous to numerous regions in the world. The extracts of this plant have been used for the treatment of a wide range of diseases including inflammation, diarrhea, sleep disorders and, moreover, for cough, analgesia. Kostic et all suggested to correlate with the antioxidant and antimicrobial (antibacterial) activity of the extracts of P. rhoeas L. from Southeast Serbia. BRIEF DESCRIPTION OF INVENTION

There are many reports about the antiviral activity of different phytochemicals against various viruses, among which bioflavonoids are noteworthy. Bioflavonoids are seen as an important natural resource in the development of antiviral drugs. Flavonoids have also been shown in a number of studies to be powerful antioxidant-anti inflammatory agent that can remove hydroxyl radicals, superoxide anions and lipid peroxyl radicals.

Flavonoids are a large group of polyphenolic substances concentrated in many plants, seeds, fruits and flowers. Among the structural components of these molecules are two benzene rings on both sides of the 3 -carbon ring. The combination of more than one hydroxyl group, sugar, oxygen and methyl group attached to these structures creates several flavonoid such as anthocyanins, flavanols, flavanones, flavones, flavan-3-ols, and isoflavones.

Papaver rhoeas red petals have been used as herbal to treat some diseases in traditional medicine. Various bioflavonoids and anthocyanins have been shown to be obtained by extraction of Papaver rhoeas red petals. There are some publications on the antibacterial, antimicrobial and antioxidant properties of these phenolic and flavonoid structures. However, the antiviral effect of bioflavanoids obtained by extraction of Papaver rhoeas red petals on SARS CoV-2 (Covid-19) and Influenza virus (H1N1, H5N1) has not been previously described.

Present invention discloses an antiviral and anti inflammatory compound is formulated with specific flavonoids which extracted from Papaver rhoeas red petals.

FIGURES

Figure 1: Quercetin (aglycone) (Formula: C15 H1007, Mass: 302.0426 g/mol)

Figure 2: Luteolin (aglycone) (Formula: C15 H10 06, Mass: 286.0476 g/mol)

Figure 3: Kaempferol (glycone), Quercitrin, Luteolin (glycone), Cynaroside (Formula: C21 H20 Oil, Mass: 448.1008 g/mol)

Figure 4: Apigenin (glycone) (Formula: C31 H34017, Mass: 678.1784 g/mol)

Figure 5: Epicatechin / Epigallocatechin (glycone-aglycone) (Formula: C37 H30 017, Mass: 746.1441 g/mol)

Figure 6: Hesperidin (aglycone)(Formula: C16 H1406, Mass: 302.0799 g/mol) DETAILED DESCRIPTION OF THE INVENTION

Petal Extraction

Classical extraction, supercritical C02 extraction and accelerated solvent extraction (ASE) methods are used. Targeted bioflavonoids is extracted from the fresh or freezed petals of Papaver rhoeas L. with ethanol-DW 1:1 mixture.

Samples are mixed in an ultrasound bath for 6 h at room temperature and in darkness during the extraction procedure. The mixture is filtered on a Buchner funnel and Whatman No. 1 filter paper. The remaining solids are washed with solvent until a clear solution is obtained. The filtrates are dried using a rotary evaporator at 30°C. The concentrate is dissolved in 0.01% Acetic acid (v/v) in DW and the solution obtained is used for further purification.

Isolation and Purification of Flavonoids a- In order to separate flavonoids, the total amount of extract crude is purified with Reverse Phase Chromatography technique in Preparative Process Chromatography (High- Performance Liquid Chromatography-HPLC). Other chromatographic separation techniques (such as normal phase, ion exchange, partition chromatography) are also be applied. b- The C-18 separation column is used for Reverse Phase Chromatography. Mobile phase consist of methanol and distilled water with gradient elution applied. The flow rate is adjusted according to the column size, length and dimensions. At the end of the reverse phase, a specific chromatogram is obtained showing the UV peak intensities of the molecular structures in the crude extract. c- The fractions of compounds are diluted with mobile phase because of elution nature. For that reason the mobile phase component is removed from original compound. Mobile phase elimination is performed with the Lyophilizator. Lyophilization is a stabilizing and drying process in which a substance is first frozen and then the quantity of the solvent is reduced. After sublimization (primary drying stage) and desorption (secondary drying stage), no further biological activation or chemical reactions occur in the purified compounds. At the end of lyophilization, the compounds also obtained as a solid substance with a high purity. d- Each purified fractions analyzed detailed to characterize and quantized the components. The confirmation and quantification of purified flavonoids are obtained by Quadrupole Time of Flight Mass Spectrometer (QTOF LC/MS). e- The specific bioflavonoids detected as a result of the HPLC and QTOF analysis are: Quercetin, Luteolin, Kaempferol, Apigenin, Quercitrin, Iso-quercetins, Cynaroside, Epicatechin, Epigallocatechin, and Hesperidin. f- These falvanoids are used in the production of antiviral-anti-inflammatory compound.

The invention is related with Quercetin, Luteolin, Kaempferol, Apigenin, Quercitrin, Iso quercetins, Cynaroside, Epicatechin, Epigallocatechin, and Hesperidin flavonoids which extracted from Papaver rhoeas' red petals. Extracted flavonoids from Papaver rhoeas' red petals are used as natural antiviral and anti-inflammatory compound. The flavonoids of the invention are in glycone (attached glycosyl group) and aglycone (lacking an attached glycosyl) form. The flavonoids are able to make covalent and coordinate bond with transition metals. Pharmaceutical composition being in the form of oral and parenteral (intramuscular - intravenous) drug composition are prepared. Pharmaceutical composition are in the form of oral food supplement. Compounds are for use as a prophylactic and therapeutic antiviral and anti-inflammatory agent in infections caused by DNA and RNA viruses. And for use as prophylactic and therapeutic antiviral and anti-inflammatory agent in infections caused by coronaviruses (SARS, MERS, Covid-19) and Influenza Viruses (H5Nl-bird flu, H1N1 -swine flu).

Quercetin [2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one]

Luteolin [2-(3,4-Dihydroxyphenyl)- 5,7-dihydroxy-4-chromenone]

Kaempferol [3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-l-benzopyran-4-one] Apigenin [5,7-Dihydroxy-2-(4-hydroxyphenyl)-4H-l-benzopyran-4-one]

Quercitrin [2-(3,4-Dihydroxyphenyl)-5,7- dihydroxy-3-[ [(2S,3R,4R,5R,6S)- 3,4,5- trihydroxy-6-methyl-2- tetrahydropyranyl]oxy]-4-chromenone] Cynaroside [2-(3,4-dihydroxyphenyl)-5-hydroxy-7-[(2S,3R,4S,5S,6R)-3,4,5 -trihydroxy-6- (hydroxymethyl)oxan-2-yl]oxychromen-4-one]

C atechin [(2R, 3 S)-2-(3 ,4-Dihy droxyphenyl)-3 ,4-dihy dro-2H-chromene-3 ,5,7 -triol]

Epigallocatechin [(2R,3R)-5, 7-dihydroxy -2-(3, 4, 5-trihydroxyphenyl)chroman-3-yl] 3,4,5- trihydroxybenzoate Hesperidin [(2S)-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-7-[(2S,3R,4S,5 S,6R)-3,4,5- trihydroxy-6-{[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxa n-2-yl]oxymethyl}oxan-2- yl] oxy-2, 3 -dihy drochromen-4-one] EXAMPLES:

Quadrupole Time of Flight Mass Spectrometer (QTOF LC/MS) data for Quercetin, Luteolin, Kaempferol, Apigenin, Quercitrin, Iso-quercetins, Cynaroside, Epicatechin, Epigallocatechin, and Hesperidin flavonoids extracted from Papaver rhoeas red petals are below.