WO2022070168A1

WO2022070168A1 - A herbal composition for treating viral infections

Info

Publication number: WO2022070168A1
Application number: PCT/IB2021/059092
Authority: WO
Inventors: Syed Mohammad UMAIR; Shaziya JAVED; Mohammad Owais; Swaleha ZUBAIR
Original assignee: Umair Syed Mohammad
Priority date: 2020-10-02
Filing date: 2021-10-04
Publication date: 2022-04-07

Abstract

The present invention provides a herbal composition for the management of microbial infections. More particularly, the present invention provides a herbal composition for the management of viral infections and the composition helps in reducing the activity of virus and prevents or manages the virus-associated diseases.

Description

“A HERBAL COMPOSITION FOR TREATING VIRAL INFECTIONS”

FIELD OF THE INVENTION

The present invention provides a herbal composition for the management of microbial infections. More particularly, the present invention provides a herbal composition for the management of viral infections.

BACKGROUND OF THE INVENTION

Coronaviruses are large, enveloped, plus-stranded RNA viruses. They cause the common cold in all age groups accounting for approximately 15% of all colds. Coronaviruses have been implicated in the etiology of gastrointestinal disease in infants. They also cause economically important diseases in animals (e.g. avian infectious bronchitis and porcine transmissible gastroenteritis). Coronaviruses get their name because in electron micrographs the envelope glycoproteins appear to form a halo or corona around the periphery of the virion. The coronaviruses are also interesting because they are the only plus-strand RNA viruses with a helical nucleocapsid. Coronaviruses are a major cause of common cold in the winter season. The virus is found throughout the world. Antibodies begin to appear in childhood, and are found in more than 90% of adults. The frequency of coronavirus respiratory infections is highly variable from year to year. The highest incidents occur in years when the cases of rhinovirus colds are lowest. Coronavirus colds tend to occur in defined outbreaks.

Coronaviruses have the largest genomes of all RNA viruses and replicate by a unique mechanism which results in a high frequency of recombination. Virions mature by budding at intracellular membranes, and coronavirus infection induces cell fusion.

Coronaviruses are transmitted by aerosols of respiratory secretions, by the fecal-oral route, and by mechanical transmission. Most viral growth occurs in epithelial cells. Occasionally the liver, kidneys, heart or eyes may be infected, as well as other cell types such as macrophages. In cold-type respiratory infections, growth appears to be localized to the epithelium of the upper respiratory tract, but there is currently no adequate animal model for the human respiratory coronaviruses. Clinically, most infections cause a mild, self-

SUBSTITUTE SHEET (RULE 26) limited disease (classical “cold” or upset stomach), but there may be rare neurological complications.

Emerging infectious diseases, such as severe acute respiratory syndrome (SARS) and Zika virus disease, present a major threat to public health. Despite intense research efforts, how, when and where new diseases appear are still a source of considerable uncertainty. A severe respiratory disease was recently reported in Wuhan, Hubei province, China.

To date, 2019-nCoV has been detected in human clinical specimens by next-generation sequencing, real-time RT-PCR, cell culture, and electron microscopy (Zhu et al., 2020). CDC recommends that clinical virology laboratories should not attempt viral isolation from specimens collected from 2019-nCoV patients under investigation (PUI). Because 2019-nCoV is a newly discovered virus, the spectrum of the available diagnostic tools is tight. At present, there are several commercially available multiplex NAAT tests for the detection of pathogenic organisms in respiratory specimens in clinical virology laboratories (Beckmann et al., 2016; Huang et al., 2018, Babady et al., 2018). They can detect HCoV-229E, -NL63, -OC43, and -HKU1. In addition, the BioFire FilmArray Respiratory Panel 2 plus and the BioFire FilmArray Pneumonia Panel plus can detect MERS-CoV in human clinical specimens.

Influenza virus is a common viral infection that can cause deadly disease in a person suffering from it, especially in high-risk groups i.e., the people who develop severe influenza viral infection. This virus is known to affect the respiratory system in children and adults, thereby leading to substantial morbidity and mortality. The people suffering from chronic disease or have weak immune systems are at high risk of developing the influenza virus in them.

Though there are several drugs and vaccines which are being tested against the pandemic caused due to the virus but a large population still doesn’t have easy reach to these expensive drugs especially in developing countries. Moreover, to successfully launch a vaccine, minimum of 20 months of time is required. Therefore, keeping in mind the real threat looming over the whole world, the present invention provides a herbal formulation modified from an age-old drug used in Unani system of medicine to ward off and to control the epidemics caused due to viruses. OBJECT OF THE INVENTION

The main object of the present invention is to provide a herbal composition for microbial infections.

Another object of the present invention is to provide a herbal composition for the management of microbial infections, particularly viral infections.

Still another object of the present invention is to provide a herbal composition which provides synergistic effect in the management of the viral infections.

Yet another object of the present invention is to provide a herbal composition which is given to reduce the activity of virus in affected subject and to prevent or manage virus- associated diseases.

SUMMARY OF THE INVENTION

The present invention provides a herbal composition for the management of microbial infections. More particularly, the present invention provides a herbal composition for the management of viral infections and the composition helps to reduce the activity of virus in affected patients and prevents or manages virus-associated diseases.

In an embodiment, the present invention provides a herbal composition for the management of viral infections comprising of Kadam or Pincushion fruit (Neolamarckia cadamba Roxb.) Mur makki or Myrrh (Commiphora myrrha), Aelwa or Aloe (Aloe vera), Habbul ghar (Paurus nobils Linn. Lourd.), Gilo neem or Giloy ( Tinospora cardifolia), Jadwar or Delphinium denudatum powder, kalonji or Black cumin (Nigella sativa) and sat e Afsanteen or Artemisia absinthium dry extract in a defined amount/ratio. The said composition is obtained from part of medicinal plants from a group comprising gum, fruits, seeds and aerial parts.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described hereinafter with reference to the accompanying drawings in which a preferred embodiment of the invention is shown. This invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough, and will fully convey the scope of the invention to those skilled in the art.

The present invention now will be described hereinafter with reference to the detailed description, in which some, but not all embodiments of the invention are indicated. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout. The present invention is described fully herein with nonlimiting embodiments and exemplary experimentation.

The present invention provides a herbal composition for the management of microbial infections. More particularly, the present invention provides a herbal composition for the management of viral infections and the composition helps in reducing the activity of virus in affected subjects and prevents or manages virus-associated diseases.

In the preferred embodiment, the present invention provides a herbal composition for the management of viral infections comprising of Kadam or Pincushion fruit (Neolamarckia cadamba Roxb.) Mur makki or Myrrh (Commiphora myrrha), Aelwa or Aloe (Aloe vera), Habbul ghar (Paurus nobils Linn. Lourd.), Gilo neem or Giloy ( Tinospora cardifolia Jadwar or Delphinium denudatum powder, kalonji or Black cumin (Nigella sativa) and sat e Afsanteen or Artemisia absinthium dry extract in a defined amount. The composition is in an oral dosage form particularly, a tablet or capsule and the composition provides synergistic effect in the management of viral infections and reduces the activity of virus as well as prevents or manages the viral infections. The novel herbal composition is composed of plants gums, resins, minerals, barks, stems and seeds in specific proportion given below in Table 1.

Table 1

Herbal composition and form of use

Neolamarckia cadamba (Roxb.) Bosser (Rubiaceae), known as Kodom in the Bengali language, is grown commonly in different parts of Bangladesh. It is an evergreen tropical tree found in Bangladesh, Nepal, India, Myanmar, Sri Lanka, the Philippines, Indonesia, and Papua New Guinea. Various parts of the plant have traditional uses as an anti-diuretic, and for the treatment of fever, anaemia and tumor. It is an herb mentioned in the Ayurvedic pharmacopoeia for the treatment of wounds, conjunctivitis, mouth ulcers, diarrhea, irritable bowel syndrome and diseases related to the urinary tract. Also, the herb is known to show anti-venom activity, anti-oxidant activity, anti-fungal, anti-bacterial activity, anti-malarial and anti-filarial activity. The fruit is used as expectorant in lung ailments and found very effective in mucoid cough. L. nobilis originates from the eastern Mediterranean and Asia Minor, where natural stands still provide a considerable part of the laurel leaf production. A chemically distinct form is native to China. L. nobilis is grown and occasionally naturalized throughout the drier tropics, subtropics and warm temperate areas. It is also cultivated as a garden and pot plant worldwide. It has expectorant, bronchial and anti-flu properties. In case of respiratory diseases, it offers a beneficial effect against flu, bronchitis, cough, respiratory tract illness.

Aelwa or Aloe (Aloe vera) is known to keep dealing properties, anti-inflammatory action, antiviral and antitumor activity, Laxative effects, Moisturizing, Antiseptic effect and antiaging effect. The majority of Aloe species occur naturally on mainland Africa, in tropical and subtropical latitudes. The genus is found almost throughout the African continent south of the Sahara Desert, except for the moist lowland forest zones and the western end of West Africa.

Tinospora cordifalia is a shrub that is native to India. Its root, stems, and leaves are used in Ayurvedic medicine. Tinospora cordifolia is used for diabetes, high cholesterol, allergic rhinitis (hay fever), upset stomach, gout, lymphoma and other cancers, rheumatoid arthritis (RA), hepatitis, peptic ulcer disease (PUD), fever, gonorrhea, syphilis, and to boost the immune system.

Nigella sativa is a small black seed that has been used for centuries in herbal medicine. The seed comes from a flowering plant (part of the Ranunculacea family) native to southwest Asia and the Mediterranean. The plant now grows throughout India, the Middle East, and Europe. It has been widely used as antihypertensive, liver tonics, diuretics, digestive, anti- diarrheal, appetite stimulant, analgesics, anti-bacterial and in skin disorders. Extensive studies on N. sativa have been carried out by various researchers and a wide spectrum of its pharmacological actions have been explored which include antidiabetic, anticancer, immunomodulator, analgesic, antimicrobial, anti-inflammatory, spasmolytic, bronchodilator, hepato-protective, renal protective, gastro-protective, antioxidant properties, etc.

Further, the primary physical virtue of white serpentine stone is to prevent headaches. It soothes tension and relaxes the mind after a confrontation, stress or anxiety. It also has recognized healing effects, both at the cardiac and digestive levels. It is also known to be antidote of snake bites and keep anti-hypertensive, antibacterial and antimicrobial properties.

Myrrh is a spiny, deciduous shrub or small tree growing about five meters tall. It usually has a small but distinct bole. An oleo-resin gum exudes from the bark of this species and various other members of the Commiphora genus. It is one of the oldest recorded medicines, having been extensively used in ancient Egypt. Myrrh is not usually cultivated, most of it being harvested from the wild. They are native to Africa, eastern Mediterranean countries, and southern Arabia. A pale yellow-white viscous liquid exudes from natural cracks or fissures in the bark or from fissures cut intentionally to harvest the material. The resin obtained from the bark of myrrh is a pungent, astringent, aromatic herb that is strongly stimulant, antiseptic and expectorant. It relieves spasms, inflammation and digestive discomfort, and encourages healing. It is particularly associated with women's health and purification rituals. The resin is taken internally in the treatment of dyspepsia, bronchial and ear infections, glandular fever, tonsillitis, pharyngitis, gingivitis, menstrual and circulatory problems. It is used in chest ailments as an expectorant in respiratory tract infections especially asthma chronic cough and also in diphtheria, tonsillitis, pharyngitis, common cold, bronchitis. It is used in chest ailments as an expectorant in respiratory tract infections especially asthma chronic cough, and also in diphtheria, tonsillitis, pharyngitis, common cold, bronchitis.

Therefore, the present invention provides a combination of an effective antiviral ingredient kadam with other ingredients helps to control the infections associated with virus infections, has great utility and represents an additional management option where, currently, effective management options are limited.

EXAMPLE 1

Viruses

Influenza (A/HINland A/H3N2) viruses were kind gift from Dr. Gunasekaran, P from King Institute of Preventive Medicine & Research, Chennai, India. The virus was cultured by nasal/throat swabs into Madin Darby Canine Kidney (MDCK) cells.

The Madin Darby Canine Kidney (MDCK) cells, obtained from the American Type Culture Collection (Manassas, VA) were used for virus inhibition assay. The cells were grown at 37°C with 5% CO₂ in Roswell Park Memorial Institute medium (RPMI; Invitrogen, No: 22400-105), supplemented with 10% foetal bovine serum (FBS; Invitrogen, No: 16140-071) and 1% Penicillin-Streptomycin (Invitrogen, No: 15140-122). The monolayers were thoroughly washed with phosphate buffered-saline (PBS, pH 7.4 at room temperature), before adding the compounds or the virus, or when quantifying the results. The various experimental setups were including appropriate cell control (cells that were not infected with the virus or treated with the plant extracts), virus control (cells that were infected only with the virus but not treated with the plant extracts in the antiviral assays), and the positive controls (virus-infected cells treated with amanatidine or Oseltamivir).

Anti-viral activity

The influenza strain A/H3N2 subtype viruses had IC 50 values 3.67 nM against the drug oseltamivir and had IC 50 values 1.32 nM against amanatidine. The other influenza strain A/H1N1 had IC 50 value 2.65 against amanatidine and IC 50 values 4.32 nM against oseltamivir respectively.

EXAMPLE 2

Protocol for Cytotoxicity studies of extract

Briefly, MDCK cells were seeded into 96-well flat-bottomed microtitre plates (Costar) at 4xl0³ cells per well. Following overnight incubation, the media of MDCK cells were aspirated, followed by addition of 100 pL of plant extract solution diluted in RPMI medium (two-fold dilutions, ranging from 0.78-100 pg/mL) and another 100 pL of growth medium (supplemented RPMI) were then added to each well. After incubation at 37°C/5% CO₂ for further 3 days, the results were quantified using 3-(4, 5-dimethylthiazol- 2-yl)-2, 5-diphenyltetrazolium bromide (MTT, Invitrogen, No: M-6494) as per the manufacturer's instructions. The optical density (OD) was measured at 540 nm using a Bio-Rad iMark TM microplate reader. The percentages of cell viability were based on the amount of living cells in compound-treated cells relative to cell controls (defined as 100% viability). Cytotoxicity graphs were then generated by plotting percentage of cell viability versus concentration of extracts. Using regression analysis of cytotoxicity curves (in Microsoft excel), a trend line that best suited the curve was selected and the corresponding equation was used to calculate 50% cytotoxic concentrations (CC50). EXAMPLE 3 In vitro micro-inhibition assay

The activity of plant extracts against influenza viruses was evaluated according to a method described elsewhere. Briefly, 96-well plates were seeded with 3x 10⁴ cells/well and incubated for 24 h at 37°C with 5% CO₂ until a confluent monolayer was attained. The cells were washed twice with PBS, and two-fold serial dilutions of plant extracts (0.78-100 pg/ml) in RPMI medium were challenged with 100 TCID50 of either of the two virus strains. To all wells, 100 pL of RPMI medium supplemented with 2 pg/mL trypsin (virus growth medium) were added. After incubation for three days at 37°C/5% CO₂, the results were quantified as previously described. The antiviral activity curve was then generated by plotting percentages of virus inhibition against concentrations of extracts. IC50, the concentration of extract essential to reduce virus-induced CPE by 50%, was expressed relative to the virus control employing dose-response curves. Using regression analysis of antiviral activity curves (in Microsoft excel), a trend line that best suited the curve was selected and the corresponding equation was used to calculate IC50 values.

EXAMPLE 4 Time-of-addition assay

The antiviral effects of extract was evaluated at different times of viral infection as described earlier. Briefly, 100 pL/well of the plant extract, serially diluted in RPMI at four concentrations (1-10 pg /mL), was added to 80% confluent MDCK cells at either 1 or 2 hours prior to infection (-1 and -2, respectively), at the time of infection (0), or 1 or 2 hours after viral infection (+1 and +2, respectively). The infection was performed by adding 100 pL/well of either H1N1 or H3N1 (100 TCID50). The various time points (-1, -2, 0, +1, +2) were tested independently in separate plates. 100 pL of virus growth medium was added to each well and the plates were then incubated for three days at 37°C/5% CO₂, after which the virus inhibition was quantified as described earlier.

EXAMPLE 5

Virus binding (atachment) assay To assess the activity of the compounds in inhibiting viral binding, an attachment assay was employed. Briefly, 80% confluent cells were chilled at 4°C for 1 hour followed by infection with 50 pL/well of H1N1 or H3N1 (200 TCID50) and simultaneous supplementation with 100 pL /well of each plant extract at four concentrations (0.78, 12.5, 25, 50 pg/ ml). All plates were held at 4°C for a further 3 h, after which the supernatant was removed; cells were washed twice with ice-cold PBS and the medium was replaced with an equal volume of RPMI and virus growth medium, and incubated for a further three days at 37°C/5% CO₂. MTT was employed to evaluate cell viability and the percentage of viral inhibition was calculated in relation to the virus control wells.

EXAMPLE 6 Penetration assay

The effect of plant extracts on viral penetration was studied according to a method described elsewhere. Briefly, 80% confluent cells were chilled at 4°C for 1 hour prior to infection with H1N1 or H3N 1 (200 TCID50) in virus growth medium and held at 4°C for further three hours. After the incubation period, specific concentrations of extracts (0.78, 12.5, 25 or 50 pg/mL) were added in triplicates to the wells with virus. The activity was studied at three time intervals (30, 60 and 120 min) employing one plate per interval at 37°C/5% CO₂. After the specified time interval, the supernatant was removed and treated with acidic PBS (pH 3) for 1 min to inactivate unpenetrated virus, and finally treated with alkaline PBS (pH 11) for neutralization. Cells were washed once with PBS (pH 7.4) and overlaid with an equal volume of RPMI and virus growth media. After three days’ incubation at 37°C/5% CO₂, cell viability was evaluated using MTT.

EXAMPLE 7

RESULTS

The IC50 of the plant extract (in DMSO) against MDCK cell line (Table 2) and (Table 3) was found out to be:

IC50 against Influenza A/H1N1 : 66±04 pg/mL

IC50 against Influenza A/H3N2 : 42±3.2 pg/Ml Table 2

The plant extract inhibited virus yield in dose dependent manner. MDCK cells were treated with DMSA or plant extract employing increasing concentration of extract followed by infection with virus (Influenza A/H1N1). Viral RNA copies in cell lysate were quantified by qRT-PCR

Table 3

The plant extract inhibited virus yield in dose dependant manner. MDCK cells were treated with DMSA or plant extract employing increasing concentration of extract followed by infection with virus (Influenza A/ A/H3N2). Viral RNA copies in cell lysate were quantified by qRT-PCR

Individual studies of individual ingredients is however known, however the novel herbal composition provided by the present invention is synergistic and exhibits effective results in treating viral infections. Therefore, the present invention provides a herbal formulation improvised from an age-old Unani drug to ward off and to control the epidemics caused due to viruses.

Many modifications and other embodiments of the invention set forth herein will readily occur to one skilled in the art to which the invention pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

CLAIMS im: A herbal composition for the management of viral infections, comprising of Kadam or Pincushion fruit (Neolamarckia cadamba Roxb.) Mur makki or Myrrh {Commiphora myrrhd), Aelwa or Aloe (Aloe vera), Habbul ghar (Paurus nobils Linn. Lourd.), Gilo neem or Giloy (Tinospora cardifolia), Jadwar or Delphinium denudatum powder, kalonji or Black cumin (Nigella sativd) and sat e Afsanteen or Artemisia absinthium dry extract in a defined amount/ratio. The herbal composition as claimed in claim 1, wherein said composition is in an oral dosage form particularly, a tablet or capsule. The herbal composition as claimed in claim 1, wherein said composition provides synergistic effect in the management of said viral infections and reduces the activity of virus as well as prevents or manages said viral infections.