DESC:FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10, rule 13)
1. Title of the invention: ORAL COMPOSITION CONTAINING NARINGIN AND
USES THEREOF
2. Applicant(s)
INDOCO REMEDIES LIMITED, an Indian company, registered under the
Indian Companies Act, 1956 having its registered office at Indoco House, 166 CST
Road, Santacruz (E), Mumbai 400 098, INDIA
3. Preamble to the description
COMPLETE SPECIFICATION
The following specification describes the invention and the manner in which it is to be
performed.
2
Field of the Invention
The present invention relates to an oral composition comprising an effective amount of
naringin, Zingiber officinale extract, and one or more other actives along with
pharmaceutically acceptable excipients. The present invention also relates to a method
for the preparation of the oral composition. 5 The present invention further relates to use
of the oral composition for the prevention or treatment of an infection caused by
coronaviruses, particularly COVID-19 caused by SARS-CoV-2 virus.
Background of the Invention
10 In the recent times, coronaviruses (CoVs) have caused severe life threatening viral
infections that have raised serious public health concerns across the world.
Coronaviruses refer to a family of enveloped, positive-sense, single-stranded, and
highly diverse RNA viruses. There are four genera (alpha, beta, gamma, and delta),
among which a-coronavirus and ß-coronavirus attract more attention because of their
15 ability to cross animal-human barriers and emerge to become major human pathogens.
To date, there have been three documented highly pathogenic and lethal CoVs, namely
Severe Acute Respiratory Syndrome (SARS-CoV), Middle East Respiratory Syndrome
(MERS-CoV) and the most recently discovered novel Severe Acute Respiratory
Syndrome-2 (SARS-CoV-2), because of their dreadful impacts on humans.
20
Unlike other types of human CoVs, SARS-CoV, MERS-CoV, and SARS-CoV-2 are
prone to infect the lower respiratory tract, resulting in acute lung injury/acute
respiratory distress syndrome (ARDS), septic shock and multi-organ failure, with high
case fatality ratio. Both SARS-CoV and MERS-CoV have higher fatality rates
25 compared to SARS-CoV-2 but the SARS-CoV-2 virus spreads rapidly through humanto-
human transmission. SARS-COV-2 is unique clinically as it is highly contagious.
The World Health Organization (WHO) named the disease caused by the SARS-CoV-2
virus as CoronaVirus Disease-19 (COVID-19). COVID-19 has ravaged across the
globe since December 2019, as it spreads faster than influenza. An infected person with
3
SARS-CoV-2 can spread the virus to 2 to 2.5 people, compared to 1.3 in influenza. As
of April 11, 2021, globally there are a total of 136 million confirmed COVID-19 cases,
with 23.6 million currently active cases. Out of the total number of cases, 109.5 million
patients have recovered and 2.9 million have died worldwide. In India alone, there have
been 13.3 million confirmed COVID-19 cases, 5 with 12 million recovered cases, 0.17
million deaths and 1.1 million active cases.
(https://www.worldometers.info/coronavirus/ accessed April 11, 2021)
The main reported clinical symptoms of COVID-19 are fever, cough and fatigue. Other
10 symptoms include sore throat, myalgia, breathlessness, loss of taste and smell. The
severity of symptoms and outcomes of COVID-19 typically vary based on the patient's
age, sex, and medical background, particularly the concurrent medical conditions.
COVID-19 progresses from mild symptoms to severe symptoms such as pneumonia.
Certain research studies suggested that uncontrolled inflammation contributes to
15 severity of COVID-19 and this is in addition to the virus damage. (Diane M. Del Valle
et al; Nature Medicine, VOL 26 October 2020 1636–1643). The elevated levels of proinflammatory
cytokines namely interleukin-6 (IL-6), interleukin-8 (IL-8), tumor
necrosis factor (TNF)-a in turn compromises the host immune response against SARSCoV-
2 infection. Preliminary results from certain observational studies suggest that
20 tocilizumab, a humanized monoclonal anti-IL-6 receptor antibody, will be beneficial in
treating severely ill COVID-19 patients. (Biran N et al; Lancet Rheumatol. 2020;2(10):
e603-e612).
Due to the rapid spread of SARS-CoV-2 virus and its high burden on health of people,
25 there have been efforts for finding effective treatment for COVID-19 through several
research projects that are ongoing at pharmaceutical companies and academic research
institutes. These initiatives are either focused on vaccines for prevention of COVID-19
or investigational drug molecules to treat COVID-19. As of March 2021, a few
therapies for COVID-19 have been approved or authorized for emergency use by
30 regulatory bodies from various countries such as the U.S. Food & Drug Administration
4
(the USFDA), European Medicines Agency (EMA), India’s Central Drugs Standard
Control Organization (CDSCO) etc. on the basis of promising results of the therapies.
(Food and Drug Administration. Coronavirus Disease 2019 (COVID-19)
www.fda.gov/emergency-preparedness-and-response/mcm-legal-regulatory-and-policyframework/
emergency-use-authorization#coviddrugs; 5 and European Medicines Agency.
Treatments and vaccines for COVID-19. www.ema.europa.eu/en/humanregulatory/
overview/public-health-threats/coronavirus-disease-covid-19/treatmentsvaccines/
treatments-vaccines-covid-19-authorised-medicines). Some of the COVID-19
therapies that have been approved for emergency use include: the antiviral remdesivir;
10 remdesivir in combination with baricitinib; dexamethasone; convalescent plasma;
bamlanivimab; and casirivimab & imdevimab (a cocktail of two monoclonal
antibodies). (Estcourt L, Roberts D. Convalescent plasma for covid-19. BMJ 2020; 370:
m3516.) However, none of the current treatment(s) offer cure for COVID-19 once
contracted.
15
Effective therapeutic interventions for the treatment and prevention of coronavirus
disease 2019 (COVID-19) are urgently needed. Intervention strategies should target
evasiveness of SARS-CoV-2 virus. One of the approaches for curbing spread of SARSCoV-
2 virus is to understand how the virus enters human cells. SARS-CoV-2 virus
20 sneaks into respiratory cells (type 2 pneumonocytes and ciliated bronchial epithelial
cells) via attachment between Angiotensin-Converting Enzyme 2 (ACE2) receptors and
Spike Glycoprotein (S Glycoprotein). The virion S-glycoprotein on the surface of
coronavirus attaches to the ACE2 receptor present on the surface of human cells. The S
glycoprotein includes two subunits, S1 and S2, and S1 determines the virus-host range
25 and cellular tropism with the key function domain, the receptor-binding domain (RBD),
while S2 mediates virus cell membrane fusion by two tandem domains, heptad repeats 1
and 2 (HR1 and HR2). After membrane fusion, the viral genome RNA is released into
the cytoplasm and replicates with the help of host cellular mechanisms. It is found that
SARS-CoV-2 enters through nose and/or mouth and thus, the COVID-19 infection
30 starts in the nose, and/or mouth. If one finds the way of blocking the virus at the initial
5
entry stage, i.e. before it spreads in the human body, moderate or severe COVID-19
infections can be prevented.
A systematic study undertaken by the present inventors lead to identification of natural
compounds that interrupt the binding of RBD 5 of the spike protein of SARS-CoV-2 to
ACE2 and the viral protease, which plays a key role in mediating viral replication and
transcription. Use of such entry point inhibitors would result in inhibition of viral
synthesis and entry respectively, thereby curbing the spread of COVID-19 infection.
10 Based on the in vitro and in vivo experimental studies of certain natural compounds, the
inventors of the present invention found naringin to have the potential for interrupting
the binding of RBD of the spike protein of SARS-CoV-2 to ACE2 and the viral
protease. Naringin, a naturally occurring flavanone glycoside, is abundantly present in
citrus fruits especially grapefruit, lemon, orange and tomatoes. The chemical name of
15 naringin is 4',5,7-trihydroxyflavanone-7-rhamnoglucoside, which is structurally
represented as follows:
It is reported in the prior art that naringin possesses antioxidant, anti-inflammatory, antiapoptotic,
anti-ulcer, anti-osteoporotic and anti-carcinogenic properties (Rui Chen et al;
20 Pharmaceutical Biology, 2016 Vol. 54, No. 12, 3203–3210). Naringin, along with other
flavonoids, is identified as having potential 2019-nCoV cell entry inhibitor through
molecular docking studies. (Bhowmik, D et al; ChemRxiv (2020), 1-27) However,
there is no report that provides supporting biological data establishing inhibitory activity
of naringin against SARS-CoV-2 virus.
25
6
Despite possessing several therapeutic properties, including the antiviral activity, there
are not many oral dosage forms of naringin available for therapeutic use as it has poor
solubility and low dissolution rate after oral administration. (Huihui Fan et al; Journal of
Pharmaceutical Development and Technology; Volume 25, 2020, Issue 5). Another
property that limits use 5 of naringin in oral dosage form is its distinct bitter taste.
A few patent and non-patent documents have reported uses and dosage forms of
naringin as summarised below:
10 PCT Int. Appl. (2020), WO2020079450 discloses pharmaceutical compositions and
their use in oral hygiene. The compositions comprise one or more flavonoids, such as
naringin and neohesperidin and polylysine and/or caprylic acid and/or a zinc salt. Such
compositions may be in the form of, for example, a solution gel, spray, chewing gum or
toothpaste suitable for use in the oral cavity.
15
US Patent No. 10213374 discloses oral care compositions comprising naringin-zinc
complexes having a 2:1 naringin to zinc molar ratio having anti-bacterial activity. The
oral care composition is provided as a toothpaste or a dentifrice, a mouthwash or a
mouth rinse, a topical oral gel or a denture cleanser.
20
US Published Patent Application US20100113373 discloses an oral antiviral
supplement composition comprising a lysine, an ascorbic compound, a flavonoid
glycoside, a threonine, and a pyridoxine. This patent application specifically teaches
oral composition in a form of powder, and tablet.
25
Chinese Published Patent Appln. CN103520010 discloses application of naringin and
tetrahydropalmatine in the production of oral care product, such as toothpaste, mouth
washing water, tooth powder, throat moistening tablet, and chewable bubble gum.
30 Su, Wei-Wei et al.; Traditional Medicine Research 2020, 5(3), 160-166, discusses the
potential application of the traditional Chinese herb Exocarpium Citri grandis (ECG) in
7
the prevention and treatment of COVID-19. It is stated in this document that the herb
ECG contains flavonoids such as naringin.
Considering the favourable anti-viral and anti-inflammatory effects of naringin, it is
desirable to pr 5 ovide oral compositions containing naringin, alone or in combination with
one or more other actives that are effective in preventing the spread of infections caused
due to coronaviruses, including the novel SARS-CoV-2 virus. Accordingly, the present
invention addresses the need for effective oral compositions.
10 Summary of the Invention
In one aspect, the present invention provides an oral composition comprising an
effective amount of each of naringin, Zingiber officinale extract, along with one or more
other actives as described herein, and pharmaceutically acceptable excipients.
15
In another aspect, the present invention provides an oral composition comprising an
effective amount of each of naringin, Zingiber officinale extract, along with one or more
other actives as described herein, and pharmaceutically acceptable excipients in a
dosage form which can be sucked or chewed by the subject in need thereof. Such
20 dosage form may be provided as lozenges, dragees, candies, pills, or chewable tablets.
In another aspect, the present invention provides an oral composition comprising an
effective amount of each of naringin, Zingiber officinale extract, along with one or more
other actives as described herein, and pharmaceutically acceptable excipients; wherein
25 the oral composition is in the form of capsules.
In another further aspect, the present invention provides a method for the preparation of
an oral composition comprising an effective amount of each of naringin, Zingiber
officinale extract, along with other actives as described herein, and pharmaceutically
30 acceptable excipients.
8
In another aspect, the present invention provides an oral composition as described
above, further comprising an additional flavonoid as described herein.
In yet another aspect, the present invention provides an oral composition comprising an
effective amount of each of naringin, Zingiber 5 officinale extract, along with one or more
other actives as described herein, and pharmaceutically acceptable excipients, for use in
the prevention or treatment of a viral infection caused by coronaviruses, wherein the
active ingredients so absorbed can act systemically to provide anti-viral and antiinflammatory
activities in addition to the relief that comes from the topical application
10 of the active ingredients to the mucous membrane of the throat.
In another further aspect, the present invention provides a method for the prevention or
treatment of a viral infection caused by coronaviruses comprising administering to the
subject an oral composition comprising an effective amount of each of naringin,
15 Zingiber officinale extract, along with other actives as described herein, and
pharmaceutically acceptable excipients, wherein the active ingredients so absorbed can
act systemically to provide anti-viral and anti-inflammatory activity in addition to the
relief that comes from the topical application of the active ingredients to the mucous
membrane of the throat.
20
Other aspects and features of the present invention will become apparent from the
following detailed description of the invention.
Detailed Description of the Invention
25
The present invention provides an oral composition comprising an effective amount of
each of naringin, Zingiber officinale extract, along with one or more other actives as
described herein, and pharmaceutically acceptable excipients.
30 The main constituent of the oral composition of the present invention is naringin.
9
As mentioned hereinabove, naringin is a naturally occurring flavanone glycoside. The
chemical name of naringin is 4',5,7- trihydroxyflavanone7-rhamnoglucoside. Naringin
is composed of naringenin, an aglycone and a disaccharide, neohesperidose attached to
the hydroxyl group at C-7. Naringin has distinctly bitter taste due to the presence of the
disaccharide, neohesperidose. Naringin’s 5 oral bioavailability is reported to be suboptimal.
Naringin is widely distributed in plant kingdom. It is found in grapefruits, (C. paradisi),
sour orange (C. aurantium), shiikuwasha (C. depressa) peel, sweet orange (C. sinensis
10 Valencia) and lemon (C. limon). (Khan M.K et al; J. Food Compos. Anal. 2014;33:85–
104) Naringin is also available from commercial sources. Naringin is recognized as
GRAS (Generally Recognized As Safe) by the USFDA (the United States Food & Drug
Administration). (https://www.fda.gov/food/food-additives-petitions/food-additivestatus-
list)
15
In addition to naringin, another key constituent of the oral composition of the present
invention is Zingiber officinale extract. Zingiber officinale is also called as “ginger” or
“sunthi” as per Ayurveda. It is reported as GRAS substance by the USFDA. It has been
indicated in the natural medicines for upper respiratory tract infections, cough,
20 bronchitis, nausea, and headache. The active part of ginger is ginger root. It includes
gingerol, gingerdione, shogaol, sesquiterpine, and monoterpene volatile oils. (Dr.
Bhavna Singh et al; World Journal of Pharma. Research; Vol 5, Issue 2, 401-409
(2016)).
25 The other actives contained in the oral composition of the present invention are selected
from one or more of vitamin C, zinc ion source, and honey.
Vitamin C is also known as ascorbic acid (L-ascorbic acid). It is a water-soluble vitamin
found in fruits and vegetables. The main sources of vitamin C are citrus fruits,
30 gooseberry, strawberries, peppers, tomatoes, cabbage, and spinach. Vitamin C is also
10
available from commercial sources. One of the most important properties of vitamin C
is that it is an antioxidant.
Zinc is naturally found in a wide variety of plant and animal foods such as beans, meat,
and fish. It is also available as a dietary 5 supplement. However, in the context of the
present invention, zinc ion source can be selected from zinc gluconate, zinc sulfate, or
zinc acetate.
Honey is a natural sweetener that comprises high amounts of reducing sugars, proteins,
10 enzymes, amino acids, minerals, polyphenols, and vitamins. Since ancient times, honey
has been used as food and as a medicine for the treatment of several serious disorders
owing to its pharmacological properties including anti-inflammatory, antioxidant,
antidiabetic, anti-cancer, antilipidemic, antifungal, and bactericidal activities. (Eteraf-
Oskouei, T. et al; Traditional and Modern Uses of Natural Honey in Human Diseases:
15 A Review. Iran. J. Basic Med. Sci. 2013, 16, 731–742.)
Thus, in an embodiment, the present invention provides an oral composition
comprising:
(a) naringin in an effective amount;
20 (b) Zingiber officinale extract in an effective amount;
(c) Vitamin C in an effective amount;
(d) zinc ion source in an effective amount;
(e) honey in an effective amount;
and one or more pharmaceutically acceptable excipients.
25
In an embodiment, the present invention provides an oral composition comprising:
(a) 20 mg to 40 mg naringin;
(b) 8 mg to 16 mg Zingiber officinale extract;
(c) 12 mg to 25 mg Vitamin C;
30 (d) 2 mg to 5 mg zinc ion source;
(e) 80 mg to 160 mg honey;
11
and one or more pharmaceutically acceptable excipients.
In an embodiment, the zinc ion source is selected from zinc gluconate, zinc sulfate, or
zinc acetate.
In 5 a preferred embodiment, the zinc ion source is zinc gluconate.
The objective of the present invention is to improve the bioavailability of the active
compound naringin by developing an oral composition or dosage form such that the
active ingredients contained therein can act systemically to provide anti-viral and anti10
inflammatory activities in addition to the relief that comes from the topical application
of the active ingredients to the mucous membrane of the throat. Thus, the objective of
the present invention is to provide an oral composition, which provides for dual
systemic and topical therapeutic effect.
15 In an embodiment, the oral composition of the present invention is provided in the form
of lozenges, dragees, chewable tablets, or chewing gums.
In another embodiment, the oral composition of the present invention is provided as
capsules comprising:
20 (a) naringin in an effective amount;
(b) Zingiber officinale extract in an effective amount;
(c) Vitamin C in an effective amount;
(d) zinc ion source in an effective amount;
(e) honey in an effective amount;
25 and one or more pharmaceutically acceptable excipients.
The oral composition of the present invention in the form of capsules may be provided
as soft-gel or hard-gel capsules.
30 In a preferred embodiment, the present invention provides lozenges comprising:
(a) naringin in an effective amount;
12
(b) Zingiber officinale extract in an effective amount;
(c) Vitamin C in an effective amount;
(d) zinc ion source in an effective amount;
(e) honey in an effective amount;
and one or more pharmaceutically 5 acceptable excipients.
In more preferred embodiment, the present invention provides lozenges comprising:
(a) 20 mg to 40 mg naringin;
(b) 8 mg to 16 mg Zingiber officinale extract;
10 (c) 12 mg to 25 mg Vitamin C;
(d) 2 mg to 5 mg zinc ion source;
(e) 80 mg to 160 mg honey;
and one or more pharmaceutically acceptable excipients.
15 In even more preferred embodiment, the present invention provides lozenges
comprising:
(a) 20 mg to 40 mg naringin;
(b) 8 mg to 16 mg Zingiber officinale extract;
(c) 12 mg to 25 mg Vitamin C;
20 (d) 2 mg to 5 mg zinc gluconate;
(e) 80 mg to 160 mg honey;
and one or more pharmaceutically acceptable excipients.
In still further preferred embodiment, the present invention provides lozenges
25 comprising:
(a) 25 mg naringin;
(b) 10 mg Zingiber officinale extract;
(c) 15 mg Vitamin C;
(d) 3 mg zinc gluconate;
30 (e) 100 mg honey;
and one or more pharmaceutically acceptable excipients.
13
The pharmaceutically acceptable excipient may be a flavouring agent or a taste-masking
agent.
In an embodiment, the flavoring 5 agents or taste-masking agent is selected from the
group consisting of thaumatin, sucralose, neotame, sodium saccharain, rebaudioside A,
steviol glycoside, licorice, glycyrrhizic acid, monoammonium glycyrrhizinate, sucrose,
glucose, fructose, maltodextrin, sorbitol, maltitol, isomalt, glycerol, vanilla flavour and
lemon flavour or a combination thereof.
10
In a specific embodiment, the present invention provides an oral composition as
described in one of the above embodiments, wherein the flavouring agent is selected
from sucralose, monoammonium glycyrrhizinate (magnasweet) and lemon flavour; or a
combination thereof.
15
As per the present invention, the oral composition as described in one of the above
embodiments contains an additional flavonoid selected from apigenin and its
derivatives, acacetin, baicalein, chrysin, luteolin, tectochrysin, kaempferol,
kaempferide, galangin, isorhamnetin, rhamnetin, myricetin, fisetin, rutin, pinobanksin,
20 pinobanksin-3-acetate, pinobanksin-7-methyl ether, pinocembrin, sakuranetin,
isosakuranetin, quercetin, hesperidin, naringin, pinostrobin and its derivatives,
trihydroxymethoxy flavanone, tetrahydroxyflavanone, ermanin, 3,5,7-trihydroxy-4'-
methoxyflavanol, 5,6,7-trihydroxy-3,4'-dimethoxyflavone, 3,7-dihydroxy-5-
methoxyflavanone, 2,5-dihydroxy-7-methoxyflavanone, 3-methylguercetin, 8-
25 methylkaempferol, or a combination thereof.
In another preferred embodiment, the additional flavonoid used in the oral composition
of the present invention is selected from quercetin, hesperidin, kaempferol, myricetin,
rutin or a combination thereof.
30
14
The oral composition of the present invention may further contain other ingredients
known for use in oral dosage forms such as acidity regulators, opacifiers, stabilising
agents, buffering agents, colouring agents, and preservatives.
In an embodiment, the 5 present invention provides a method for the preparation of the
oral composition comprising an effective amount of naringin; Zingiber officinale
extract; Vitamin C; zinc ion source; honey; and one or more pharmaceutically
acceptable excipients.
10 In a preferred embodiment, the present invention provides a method for the preparation
of lozenges comprising:
(i) mixing sugar and water, and bringing it to boil to dissolve the mixture to obtain
a viscous mass;
(ii) heating the above viscous mass at 140°C, and passing it through vapo
15 separation chamber;
(iii) adding the actives such as naringin, Zingiber officinale extract; Vitamin C; zinc
ion source; honey to the mixture obtained in step (ii) above; and immediately
starting drawing ribbon with cooling;
(iv) adding flavouring agents during ribboning and cooling; and
20 (v) rolling, molding as lozenges, and drying.
The lozenges provided as per the present invention has pleasant taste as the inventors
have been successful in masking the bitter taste of naringin.
25 The oral compositions of the present invention when provided in the form of capsules,
the method for the preparation of capsules may involve admixing or otherwise
combining the above-described actives and other pharmaceutically acceptable
excipients using, for example, methods and equipment known by those of skill in the
art, followed by adding the admixture empty gelatin capsules.
30
15
The dosage range of the oral composition of the present invention, including the
lozenges, per day can vary widely depending upon the patient’s age, severity of
symptoms of coronavirus infections, and any other medical condition that the patient is
suffering from.
5
When the oral composition is provided in the form of lozenges, typically, the dose will
be one to two lozenge administered 4 to 5 times a day.
As mentioned herein above, the present inventors have tested the activity of naringin
10 against the SARS-CoV-2 virus through in vitro and in vivo experimentation, and
naringin is found to be effective against the SARS-CoV-2 virus by showing an
inhibition of the virus replication. Particularly, naringin exhibited prophylactic and
therapeutic efficacy in Human Corona virus-229E infected mice model.
Adding Zingiber officinale extract, Vitamin C, zinc ion source and honey to naringin,
15 supplements the prophylactic and therapeutic efficacy of naringin when provided in the
form of an oral composition such as lozenges for use in the prevention or treatment of
viral infections caused due to coronaviruses, including COVID-19, which is caused by
the SARS-CoV-2 virus.
Accordingly, in an embodiment the present invention provides the oral composition
20 comprising an effective amount of each of naringin; Zingiber officinale extract; Vitamin
C; zinc ion source; honey; and one or more pharmaceutically acceptable excipients for
use in the prevention or treatment of viral infections caused due to coronaviruses.
In another embodiment, the present invention provides the oral composition comprising
25 an effective amount of each of naringin; Zingiber officinale extract; Vitamin C; zinc ion
source; honey; and one or more pharmaceutically acceptable excipients for use in the
prevention or treatment of COVID-19 caused by SARS-CoV-2 virus.
In an embodiment, the present invention provides capsules comprising an effective
30 amount of each of naringin; Zingiber officinale extract; Vitamin C; zinc ion source;
16
honey; and one or more pharmaceutically acceptable excipients for use in the prevention
or treatment of COVID-19 caused by SARS-CoV-2 virus.
In another embodiment, the present invention provides capsules comprising an effective
amount of each of naringin; Zingiber 5 officinale extract; Vitamin C; zinc ion source;
honey; and one or more pharmaceutically acceptable excipients for use in the
prevention or treatment of viral infections caused due to coronaviruses.
In a preferred embodiment, the present invention provides lozenges comprising an
10 effective amount of each of naringin; Zingiber officinale extract; Vitamin C; zinc ion
source; honey; and one or more pharmaceutically acceptable excipients for use in the
prevention or treatment of viral infections caused due to coronaviruses.
In another preferred embodiment, the present invention provides lozenges comprising
15 an effective amount of naringin; Zingiber officinale extract; Vitamin C; zinc ion source;
honey; and one or more pharmaceutically acceptable excipients for use in the prevention
or treatment of COVID-19 caused by SARS-CoV-2 virus.
In a preferred embodiment, the present invention provides lozenges comprising:
(a) 20 mg to 40 mg naringin;
20 (b) 8 mg to 16 mg Zingiber officinale extract;
(c) 12 mg to 25 mg Vitamin C;
(d) 2 mg to 5 mg zinc gluconate;
(e) 80 mg to 160 mg honey;
and one or more pharmaceutically acceptable excipients; for use in the prevention or
25 treatment of viral infections caused due to coronaviruses.
In another preferred embodiment, the present invention provides lozenges comprising:
(a) 20 mg to 40 mg naringin;
(b) 8 mg to 16 mg Zingiber officinale extract;
(c) 12 mg to 25 mg Vitamin C;
17
(d) 2 mg to 5 mg zinc gluconate;
(e) 80 mg to 160 mg honey;
and one or more pharmaceutically acceptable excipients; for use in the prevention or
treatment of COVID-19 caused by SARS-CoV-2 virus.
In a more preferred embodiment, 5 the present invention provides lozenges comprising:
(a) 25 mg naringin;
(b) 10 mg Zingiber officinale extract;
(c) 15 mg Vitamin C;
(d) 3 mg zinc gluconate;
10 (e) 100 mg honey;
and one or more pharmaceutically acceptable excipients; for use in the prevention or
treatment of viral infections caused due to coronaviruses.
In an even more preferred embodiment, the present invention provides lozenges
comprising:
15 (a) 25 mg naringin;
(b) 10 mg Zingiber officinale extract;
(c) 15 mg Vitamin C;
(d) 3 mg zinc gluconate;
(e) 100 mg honey;
20 and one or more pharmaceutically acceptable excipients; for use in the prevention or
treatment of COVID-19 caused by SARS-CoV-2 virus.
In an embodiment, the present invention provides a method for the prevention or
treatment of a viral infection caused by coronaviruses comprising administering to a
25 subject in need thereof an oral composition comprising an effective amount of naringin;
Zingiber officinale extract; Vitamin C; zinc ion source; honey; and one or more
pharmaceutically acceptable excipients.
18
In another embodiment, the present invention provides a method for the prevention or
treatment of COVID-19 caused by SARS-CoV-2 virus comprising administering to a
subject in need thereof an oral composition comprising an effective amount of naringin;
Zingiber officinale extract; Vitamin C; zinc ion source; honey; and one or more
pharmaceutically 5 acceptable excipients.
In a preferred embodiment, the present invention provides a method for the prevention
or treatment of a viral infection caused by coronaviruses comprising administering to a
subject in need thereof, lozenges comprising:
10 (a) 20 mg to 40 mg naringin;
(b) 8 mg to 16 mg Zingiber officinale extract;
(c) 12 mg to 25 mg Vitamin C;
(d) 2 mg to 5 mg zinc gluconate;
(e) 80 mg to 160 mg honey;
15 and one or more pharmaceutically acceptable excipients.
In another preferred embodiment, the present invention provides a method for the
prevention or treatment of COVID-19 caused by SARS-CoV-2 virus comprising
administering to a subject in need thereof, lozenges comprising:
(a) 20 mg to 40 mg naringin;
20 (b) 8 mg to 16 mg Zingiber officinale extract;
(c) 12 mg to 25 mg Vitamin C;
(d) 2 mg to 5 mg zinc gluconate;
(e) 80 mg to 160 mg honey;
and one or more pharmaceutically acceptable excipients.
25 In a more preferred embodiment, the present invention provides a method for the
prevention or treatment of a viral infection caused by coronaviruses comprising
administering to a subject in need thereof, lozenges comprising:
19
(a) 25 mg naringin;
(b) 10 mg Zingiber officinale extract;
(c) 15 mg Vitamin C;
(d) 3 mg zinc gluconate;
5 (e) 100 mg honey;
and one or more pharmaceutically acceptable excipients.
In an even more preferred embodiment, the present invention provides a method for the
prevention or treatment of COVID-19 caused by SARS-CoV-2 virus comprising
administering to a subject in need thereof, lozenges comprising:
10 (a) 25 mg naringin;
(b) 10 mg Zingiber officinale extract;
(c) 15 mg Vitamin C;
(d) 3 mg zinc gluconate;
(e) 100 mg honey;
15 and one or more pharmaceutically acceptable excipients.
The general terms used hereinbefore and hereinafter preferably have within the context
of this disclosure the following meanings, unless otherwise indicated. Thus, the
definitions of the general terms as used in the context of the present invention are
provided herein below:
20 The singular forms "a," "an," and "the" include plural reference unless the context
clearly dictates otherwise.
The term "effective amount", as used herein, refers to an amount of each of the actives
namely naringin, Zingiber officinale extract; Vitamin C, zinc ion source and honey in an
amount that is capable of performing the intended result in the prevention or treatment
25 of a viral infection caused by coronaviruses, including COVID-19 caused by SARSCoV-
2 virus.
20
The term “prevention”, as used herein, refers to preventive measures, wherein the aim is
to prevent or lessen the chances or risk of an undesired affliction, such as to prevent
occurrence, development and progression of viral infection, and conditions associated
with or resulting from the infections that are caused by coronaviruses, including
5 COVID-19 caused by SARS-CoV-2 virus.
The term “treatment” as used herein, administration of oral composition of the present
invention to a subject or a patient, who has an infection caused by coronaviruses,
including SARS-CoV-2 virus, or a symptom of the infection or a predisposition towards
the infection, with the purpose to heal, alleviate, relieve, remedy, ameliorate, improve or
10 affect the infection, the symptoms of the infection or the predisposition towards the
infection.
The term “subject”, as used herein, means a human being, who may be of an adult age
or a child of above 12 years of age, who is in need of prevention or treatment of viral
infections caused due to coronaviruses, including COVID-19 caused by SARS-CoV-2
15 virus.
The term "bioavailability" refers to the rate and extent to which the active ingredient or
active moiety is absorbed from a drug product and becomes available at the site of
action. (Pharmaceutical Research, 2001, 18, 12, 1645-1650).
The term “lozenge” as used herein includes all formulations in which the product is
20 formed by cooling a molten mass of sugar-base or sugar alcohol base comprising
effective amount of Naringin, Zingiber officinale extract and one or more other actives
as described herein. The pharmaceutical lozenge composition of the invention possesses
good feel and good taste in the mouth.
The term "dragee" refers to any solid or semi-solid core and an inert sugar coating
25 around the core where at least a majority of the substrate is designed to dissolve in an
oral cavity. The term "dragee" covers a range of confectioneries, the common
denominator is a structure with a solid core or firm coated by a hard candy.
21
The term “Chewable tablets” refers to a solid dosage form which can be taken by mouth
and are designed to be processed by chewing to facilitate release of the active
ingredient(s) rather than to be swallowed whole.
The following examples are meant to illustrate the present invention and should not be
5 construed as a limitation of its scope.
Example 1:
The following table illustrates the composition of the currently preferred oral
composition of the present invention i.e. lozenges:
Sr.
No
Ingredients Quantity per lozenges
(in mg)
1 Naringin 25
2 Zingiber officinale
extract (Sunthi)
10
3 Vitamin C 15
4 Zinc gluconate 3
5 Natural honey 100
6 Flavour - Lemon 2.5
7 Monoammonium
glycyrrhizinate
(magnasweet or MM100)
5.0
8 Sucralose 3.2
9 Colour (Dark brown) 0.6
Method for the preparation of the lozenges:
10 (i) Mixing sugar and water, and bringing it to boil to dissolve the mixture to obtain
a viscous mass;
(ii) heating the above viscous mass at 140°C, and passing it through vapo separation
chamber;
(iii) adding the actives: naringin, Zingiber officinale extract; Vitamin C; zinc ion
15 source; honey to the mixture obtained in step (ii) above; and immediately start
drawing ribbon with cooling;
(iv) adding flavouring agents during ribboning and cooling;
(v) rolling, molding as lozenges, and drying.
22
Example 2:
In vitro study of Naringin against SARS-CoV2 virus:
The objective of this study was to evaluate the prophylactic and therapeutic anti-viral
activity of Naringin, the key active of the oral composition of the present invention
5 A) Test method:
The test is carried out in the following two stages:
1. Determination of the 50% cytotoxic concentration (CC50) and Maximum Non
Toxic Dose (MNTD) for Naringin
2. In vitro anti-viral assessment of Naringin is done in two modes:
10 I. Prophylactic: Vero cells treated with Naringin prior to SARS-CoV-2
virus infection
II. Therapeutic: Vero cells infected with SARS-CoV-2 virus followed
by treatment with Naringin.
B) Test virus summary:
Realm Riboviria
Order Nidovirales
Family Coronaviridae
Genus Betacoronavirus
Species COVID-19
NCBI Accession number
for virus isolate
MT416726
15
C) Experimental conditions:
Host Cell line used for testing Vero cell line
Virus used for testing COVID 19 (SARS-CoV-2) at 0.1 MOI
Test Controls
? CC50 determination 400 µM Formaldehyde
0.72 µM Hydroxychloroquine Sulfate
23
? EC50 determination
Diluent used for the assay 2% Minimum Essential Media (MEM)
Incubation Period
? CC50 determination
? EC50 determination
72 hours post drug treatment
72 hours post virus infection
Incubation conditions 37 ºC, 5 % CO2
D) Test data:
The antiviral analysis of Naringin using the above test method under the afore
specified conditions indicated that the compound has 72.7 µM and 72.1 µM EC50
values at 4 hr and 8 hr respectively 5 when the cells were treated with Naringin after
SARS-CoV-2 virus infection.
Example 3:
In vivo study: Evaluation of prophylactic efficacy of Naringin (Compound N) in
10 Human Corona virus-229E infected mice model
The objective of this study was to evaluate the prophylactic anti-viral activity of
Naringin, the key active of the oral composition of the present invention, in human
Corona virus-229E (HCoV-229E) infected mice model.
I. Mice infection and Treatment Protocol:
15 Mice and treatment: Mice (female BALB/c, 6–8 weeks old, n=9), weighing 22–25
g, are sorted into 10 groups (10 groups- 1 disease control group, 1 normal control
and 8 treatment groups) with each group constituting 9 animals. Mice in
respective pre-designated groups will be pre-administered with test products at 30
mins before virus challenge and 4 hrs before challenge with a desired
20 concentration through aforementioned routes everyday up to 7 days.
24
Infection and Analysis: Mice will be challenged with HCoV-229E virus intranasally
after 30 mins and 4 hrs of drug dosage on day 1 in pre-designated groups.
The infection will be confirmed at 24 hpi (hour post-infection) from disease
control group. The drug dosage will be continued up to 7th day at a determined
dose. The decrease in viral 5 load will be assessed by qPCR (quantitative
polymerase chain reaction) at the end of the study.
Protocol day Description
Activity Days Normal and
Disease Control
Naringin test
sample
Dosage
(Intranasal
and Oral)
Day 1
(30 mins
before
challenge)
Saline 300 µg/mice
Day 1
(4 Hr before
challenge)
Saline 300 µg/mice
Virus
challenge
Day 1 Saline Infect with
HCOV 229E (106
TCID50 dose) -
intranasally
Dosage
(Intranasal
and Oral)
Day 2 to Day
6
Saline 300 µg/mice
Final bleed
and sacrifice
Day 7 Collection of samples for Viral load
estimation and cytokines
The qPCR analysis of lung tissues at the end of the study suggest that the group
treated with a naringin test sample, 30 mins before virus challenge has shown
10 better reduction in viral load in lungs with relative expression of 0.2774 (72.26%
reduction) and 0.3410 (65.9% reduction) in the group treated 4 hrs before virus
challenge compared to disease control group with relative expression of 1.
? Quantification of serum cytokines:
15 Background: A rapid and coordinated immune response during COVID-19
infection leads to enhanced secretion of various cytokines, which acts as a defense
mechanism against the virus. Numerous reports suggest that individuals affected
25
with SARS-CoV-2 have dysregulated cytokine production from both innate and
adaptive immune cells. In the case of SARS-CoV-2, infected hematopoietic cell,
monocyte-macrophages, and other immune cells trigger enhanced secretion of
pro-inflammatory cytokines like IL-6, IL-10, and TNF-a.
5
? Mouse IL-6 ELISA: Quantification of serum IL-6
I. Reagent Preparation:
1. Wash Buffer (20X):
Dilution: To make wash buffer (1X), add 5ml of wash buffer (20X) to 95ml
10 of DI water. This is the working solution.
2. Assay Diluent (5X):
Dilution: To make assay diluent (1X), add 10ml of assay diluent (5X) to
40ml of DI water. This is the working solution.
3. Biotin Conjugated Detection Antibody (78µl):
15 Dilution: Add 40µl of detection antibody to 4960µl of assay diluent (1X) to
make final volume to 5ml.
4. Concentrated Streptavidin-HRP (78 µl):
Diluent: Add 40µl of concentrated streptavidin-HRP (horseradish
peroxidase (HRP) conjugated streptavidin) to 4960 µl of assay diluent (1X)
20 to make final volume to 5ml.
5. Standard (Recombinant Mouse IL-6, Lyophilized):
Reconstitution: Reconstitute lyophilized Mouse IL-6 standard with 650 µl of
distilled water to achieve final concentration 500 pg/ml. Mix gently and
leave to stand for 15 mins before further dilution. Use immediately and do
25 not store reconstituted standard.
26
II. Assay Procedure:
1. Bring all reagents to room temperature prior to use. A standard curve is
required for each assay.
2. Add 100 µl/well of Standards and Samples to the plate. Perform two-fold
serial dilutions of the 250 5 pg/ml top standard, either within the plate or in
separate tubes. Thus, the Mouse IL-6 standard concentrations are 250 pg/ml,
125 pg/ml, 62.5 pg/ml, 31.25 pg/ml, 15.6 pg/ml 7.8pg/ml and 0pg/ml. Assay
diluent (1X) serves as the zero standard (0 pg/ml). Seal plate and incubate at
37°C for 2 hours.
10 3. Aspirate and wash plate 4 times with Wash Buffer (1X) and blot residual
buffer by firmly tapping plate upside down on absorbent paper. Wipe of any
liquid from the bottom outside of the microtiter wells as any residue can
interfere in the reading step. All the washes should be performed similarly.
4. Add 100 µl of diluted Detection Antibody solution to each well, seal plate
15 and incubate at 37°C for 1 hour.
5. Wash plate 4 times with Wash Buffer (1X) as in step 3.
6. Add 100 µl of diluted Streptavidin-HRP solution to each well, seal plate and
incubate at 37°C for 30 minutes.
7. Wash plate 4 times with Wash Buffer (1X) as in step 3. For this final wash,
20 soak wells in Wash buffer for 30 seconds to 1 minute for each wash. This
will help minimize background.
8. Add 100µl of TMB Substrate solution and incubate in the dark for 15
minutes 37°C. Positive wells should turn bluish in color. It is not necessary
to seal the plate during this step.
25 9. Stop reaction by adding 100µl of Stop Solution to each well. Positive wells
should turn from blue to yellow.
27
10. Read the absorbance at 450 nm within 30 minutes of stopping reaction.
Results:
The results of this study demonstrated reduction in IL-6 levels (P<0.001) in the
treatment group compared to the disease control group, wherein the mice were
treated with naringin test sample prior 5 to the infection. The group in which the
mice that were treated with 300 µg/mice of naringin test sample 30 minutes before
the infection showed significant reduction in serum IL-6 levels to the value of
42.23± 1.84 pg/ml compared to the disease control group (167.03 ± 7.89 pg/ml).
In another treatment group of mice wherein the mice were treated with another
10 naringin test sample 4 hours before the infection showed significant reduction in
serum IL-6 levels of the value to 42.23± 1.84 pg/ml compared to the disease
control group (167.03 ± 7.89 pg/ml).
? Mouse IL-10 ELISA: Quantification of serum IL-10
I. Reagent Preparation:
15 1. Wash Buffer (20X):
Dilution: To make wash buffer (1X), add 5 ml of wash buffer (20X) to 95
ml of DI water. This is the working solution.
2. Assay Diluent (5X):
Dilution: To make assay diluent (1X), add 10ml of assay diluent (5X) to 40
20 ml of DI water. This is the working solution.
3. Biotin Conjugated Detection Antibody (110µl):
Dilution: Add 52.08 µl of detection antibody to 4947.92µl of assay diluent
(1X) to make final volume to 5 ml.
4. Concentrated Streptavidin-HRP (210 µl):
25 Diluent: Add 100µl of concentrated streptavidin-HRP to 4900µl of assay
diluent (1X) to make final volume to 5 ml.
28
5. Standard (Recombinant Mouse IL-10, 0.5 µg/ml; 10 µl per vial):
Dilution: Thaw and dilute the recombinant protein by adding 2 µl of
standard solution in 998 µl of Assay diluent (1X) to prepare 1000 µl of top
standard (1000pg/ml).
5 II. Assay Procedure:
1. Bring all reagents to room temperature prior to use. A standard curve is
required for each assay.
2. Add 100µl/well of Standards and Samples to the plate. Perform two-fold
serial dilutions of the 1000pg/ml top standard, either within the plate or in
10 separate tubes. Thus, the Mouse IL-10 standard concentrations are
1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.25pg/ml,
15.6pg/ml 7.8pg/ml and 0pg/ml. Assay diluent (1X) serves as the zero
standard (0pg/ml). Seal plate and incubate at 37°C for 2 hours.
3. Aspirate and wash plate 4 times with Wash Buffer (1X) and blot residual
15 buffer by firmly tapping plate upside down on absorbent paper. Wipe of any
liquid from the bottom outside of the microtiter wells as any residue can
interfere in the reading step. All the washes should be performed similarly.
4. Add 100µl of diluted Detection Antibody solution to each well, seal plate
and incubate at 37°C for 1 hour.
20 5. Wash plate 4 times with Wash Buffer (1X) as in step 3.
6. Add 100 µl of diluted Streptavidin-HRP solution to each well, seal plate and
incubate at 37°C for 30 minutes.
7. Wash plate 4 times with Wash Buffer (1X) as in step 3. For this final wash,
soak wells in Wash buffer for 30 seconds to 1 minute for each wash. This
25 will help minimize background.
29
8. Add 100µl of TMB Substrate solution and incubate in the dark for 15
minutes 37°C. Positive wells should turn bluish in color. It is not necessary
to seal the plate during this step.
9. Stop reaction by adding 100µl of Stop Solution to each well. Positive wells
5 should turn from blue to yellow.
10. Read the absorbance at 450 nm within 30 minutes of stopping reaction.
Results:
Interleukin 10 (IL-10) levels were significantly elevated in the HCOV disease
control indicating a cytokine storm. In the treatment groups, there was a
10 statistically significant decrease in IL-10 levels (P<0.001).
? Mouse TNF-a ELISA: Quantification of TNF-a
I. Reagent Preparation:
1. Wash Buffer (20X):
Dilution: To make wash buffer (1X), add 5ml of wash buffer (20X) to 95ml
15 of DI water. This is the working solution.
2. Assay Diluent (5X):
Dilution: To make assay diluent (1X), add 10 ml of assay diluent (5X) to 40
ml of DI water. This is the working solution.
3. Biotin Conjugated Detection Antibody (100µl):
20 Dilution: Add 50 µl of detection antibody to 5950 µl of assay diluent (1X) to
make final volume to 6 ml.
4. Concentrated Streptavidin-HRP (240 µl):
Diluent: Add 83.33µl of concentrated streptavidin-HRP to 4916.67µl of
assay diluent (1X) to make final volume to 5ml.
30
5. Standard (Mouse TNF-a; Lyophilized):
Reconstitution: Reconstitute lyophilized Mouse TNF-a standard with 50 µl
of Distilled water to achieve final concentration 0.5 µg/ml. Mix gently and
leave to stand for 15 mins before further dilution. Use immediately, and do
5 not store reconstituted standard.
II. Assay Procedure:
1. Bring all reagents to room temperature prior to use. A standard curve is
required for each assay.
2. Add 100µl/well of Standards and Samples to the plate. Perform six two-fold
10 serial dilutions of the 1000pg/ml top standard, either within the plate or in
separate tubes. Thus, the Mouse TNF-a standard concentrations are
1000pg/ml, 450pg/ml, 225pg/ml, 112.5pg/ml, 56.25pg/ml, 28.13pg/ml,
14.06pg/ml 7.03pg/ml, 3.52pg/ml and 0pg/ml. Assay diluent (1X) serves as
the zero standard (0pg/ml). Seal plate and incubate at 37°C for 2 hours.
15 3. Aspirate and wash plate 4 times with Wash Buffer (1X) and blot residual
buffer by firmly tapping plate upside down on absorbent paper. Wipe of any
liquid from the bottom outside of the microtiter wells as any residue can
interfere in the reading step. All the washes should be performed similarly.
4. Add 100µl of diluted Detection Antibody solution to each well, seal plate
20 and incubate at 37°C for 1 hour.
5. Wash plate 4 times with Wash Buffer (1X) as in step 3.
6. Add 100µl of diluted Streptavidin-HRP solution to each well, seal plate and
incubate at 37°C for 30 minutes.
7. Wash plate 4 times with Wash Buffer (1X) as in step 3. For this final wash,
25 soak wells in Wash buffer for 30 seconds to 1 minute for each wash. This
will help minimize background.
31
8. Add 100µl of TMB Substrate solution and incubate in the dark for 15
minutes 37°C. Positive wells should turn bluish in color. It is not necessary
to seal the plate during this step.
9. Stop reaction by adding 100µl of Stop Solution to each well. Positive wells
5 should turn from blue to yellow.
10. Read the absorbance at 450 nm within 30 minutes of stopping reaction.
Results:
The results of this study demonstrated reduction in TNF-? levels (P<0.001) in the
10 treatment group compared to the disease control group (55.86 ± 3.23 pg/ml),
wherein the mice were treated with naringin test sample prior to the infection.
The group in which the mice that were treated with 300 µg/mice of naringin test
sample 30 minutes before the infection showed significant reduction in TNF-?
levels to the value of 7.95±0.28 pg/ml compared to the disease control group
15 (55.86 ± 3.23 pg/ml). In another treatment group of mice wherein the mice were
treated with another naringin test sample 4 hours before the infection, significant
reduction in TNF-? levels to the value of 19.3 ± 1.83 pg/ml was observed
compared to the disease control group (55.86 ± 3.23 pg/ml).
Example 4:
20 In vivo study: Evaluation of therapeutic efficacy of Naringin in Human Corona
virus-229E infected mice model
The objective of this study was to evaluate the therapeutic anti-viral activity of
Naringin, the key active of the oral composition of the present invention, in human
Corona virus-229E (HCoV-229E) infected mice model.
25 ? Mice infection and Treatment Protocol:
Mice (female BALB/c, 6–8 weeks old, n=9), weighing 20-22 g, are sorted into 11
groups (11 groups- 1 disease control group, 1 normal control group, 1 positive
control group and 8 treatment groups) with each group constituting 9 animals.
32
Mice in respective predesignated groups is challenged with HCoV-229E virus
intranasally and incubation is carried out for 6 hrs.
Post viral challenge, at 6 hpi (hour post-infection) animals are administered with
test samples of naringin at desired concentrations through pre-determined route of
administration. The test sample treatment is continued 5 for 7 days. The infection is
confirmed at 24 hpi (hour post-infection) in the disease control group and the
reduction in viral load is determined in all the study groups at the end of the study
by qPCR (quantitative polymerase chain reaction) method using lung tissues.
Treatment
Protocol day Description
Virus
Infection
Day 0
Infect with HCOV 229E (TCID 50 dose) – intranasally
(TCID 50 dose - 50% Tissue Culture Infective Dose
per ml)
Treatment Control Low dose High dose
1st Dose 6 hpi (hour
postinfection)
Saline Pre-determined
concentration of
test samples
Pre-determined
concentration of
test samples
Dosage
frequency
Day 1 – Day
7
Saline
Final
bleed and
sacrifice
Day 7 Collection of samples for viral load estimation and
Cytokine analysis
The qPCR analysis of lung tissues from various treatment groups was carried out
10 to evaluate the viral load at the end of the study. The results suggest that the
groups treated with a test sample of Naringin at a concentration of 150 µg/mice
(low dose) and 300 µg/mice (high dose) showed up to 92.32% and 96.21%
reduction of viral load with relative expression of 0.077 and 0.038.
? Quantification of serum cytokines:
15 Background: A rapid and coordinated immune response during COVID-19
infection leads to enhanced secretion of various cytokines, which acts as a defense
mechanism against the virus. Numerous reports suggest that individuals affected
33
with SARS-CoV-2 have dysregulated cytokine production from both innate and
adaptive immune cells. In the case of SARS-CoV-2, infected hematopoietic cell,
monocyte-macrophages, and other immune cells trigger enhanced secretion of
pro-inflammatory cytokines like IL-6, IL-10, and TNF-a.
? Mouse IL-5 6 ELISA: Quantification of serum IL-6
I. Reagent Preparation:
1. Wash Buffer (20X):
Dilution: To make wash buffer (1X), add 5 ml of wash buffer (20X) to 95
ml of DI water. This is the working solution.
10 2. Assay Diluent (5X):
Dilution: To make assay diluent (1X), add 10ml of assay diluent (5X) to 40
ml of DI water. This is the working solution.
3. Biotin Conjugated Detection Antibody (78 µl):
Dilution: Add 40 µl of detection antibody to 4960 µl of assay diluent (1X) to
15 make final volume to 5ml.
4. Concentrated Streptavidin-HRP (78 µl):
Diluent: Add 40µl of concentrated streptavidin-HRP (horseradish
peroxidase (HRP) conjugated streptavidin) to 4960 µl of assay diluent (1X)
to make final volume to 5 ml.
20 5. Standard (Recombinant Mouse IL-6, Lyophilized):
Reconstitution: reconstitute lyophilized Mouse IL-6 standard with 650 µl of
Distilled water to achieve final concentration 500 pg/ml. Mix gently and
leave to stand for 15 mins before further dilution. Use immediately, and do
not store reconstituted standard.
25 II. Assay Procedure
34
1. Bring all reagents to room temperature prior to use. A standard curve is
required for each assay.
2. Add 100 µl/well of Standards and Samples to the plate. Perform two-fold
serial dilutions of the 250 pg/ml top standard, either within the plate or in
separate tubes. Thus, the Mouse IL-5 6 standard concentrations are 250 pg/ml,
125 pg/ml, 62.5 pg/ml, 31.25 pg/ml, 15.6 pg/ml 7.8 pg/ml and 0 pg/ml.
Assay diluent (1X) serves as the zero standard (0 pg/ml). Seal plate and
incubate at 37°C for 2 hours.
3. Aspirate and wash plate 4 times with Wash Buffer (1X) and blot residual
10 buffer by firmly tapping plate upside down on absorbent paper. Wipe of any
liquid from the bottom outside of the microtiter wells as any residue can
interfere in the reading step. All the washes should be performed similarly.
4. Add 100 µl of diluted Detection Antibody solution to each well, seal plate
and incubate at 37°C for 1 hour.
15 5. Wash plate 4 times with Wash Buffer (1X) as in step 3.
6. Add 100µl of diluted Streptavidin-HRP solution to each well, seal plate and
incubate at 37°C for 30 minutes.
7. Wash plate 4 times with Wash Buffer (1X) as in step 3. For this final wash,
soak wells in Wash buffer for 30 seconds to 1 minute for each wash. This
20 will help minimize background.
8. Add 100µl of TMB Substrate solution and incubate in the dark for 15
minutes 37°C. Positive wells should turn bluish in color. It is not necessary
to seal the plate during this step.
9. Stop reaction by adding 100 µl of Stop Solution to each well. Positive wells
25 should turn from blue to yellow.
10. Read the absorbance at 450 nm within 30 minutes of stopping reaction.
35
Results:
The results of this study demonstrated significant reduction in IL-6 levels
(P<0.001) in the treatment group compared to the disease control group. The
group in which the infected mice were treated with 150µg/mice (low dose) and
300 µg/mice (high dose) of a Naringin test 5 sample showed significant reduction to
90.14 ± 2.74 pg/ml (with low dose) and 60.87 ± 5.54 pg/ml (with high dose) in
serum IL-6 levels compared to the disease control group (201.16 ± 10.63 pg/ml).
? Mouse IL-10 ELISA: Quantification of serum IL-10
I. Reagent Preparation
10 1. Wash Buffer (20X):
Dilution: To make wash buffer (1X), add 5ml of wash buffer (20X) to 95ml
of DI water. This is the working solution.
2. Assay Diluent (5X):
Dilution: To make assay diluent (1X), add 10 ml of assay diluent (5X) to
15 40ml of DI water. This is the working solution.
3. Biotin Conjugated Detection Antibody (110 µl):
Dilution: Add 52.08µl of detection antibody to 4947.92µl of assay diluent
(1X) to make final volume to 5ml.
4. Concentrated Streptavidin-HRP (210 µl):
20 Diluent: Add 100µl of concentrated streptavidin-HRP to 4900µl of assay
diluent (1X) to make final volume to 5ml.
5. Standard (Recombinant Mouse IL-10, 0.5 µg/ml; 10µl per vial):
Dilution: Thaw and dilute the recombinant protein by adding 2µl of standard
solution in 998µl of Assay diluent (1X) to prepare 1000µl of top standard
25 (1000pg/ml).
II. Assay Procedure
36
1. Bring all reagents to room temperature prior to their use. A standard curve is
required for each assay.
2. Add 100 µl/well of Standards and Samples to the plate. Perform two-fold
serial dilutions of the 1000 pg/ml top standard, either within the plate or in
separate tubes. Thus, the Mouse IL-5 10 standard concentrations are 1000
pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml, 62.5 pg/ml, 31.25 pg/ml, 15.6
pg/ml 7.8 pg/ml and 0 pg/ml. Assay diluent (1X) serves as the zero standard
(0pg/ml). Seal plate and incubate at 37°C for 2 hours.
3. Aspirate and wash plate 4 times with Wash Buffer (1X) and blot residual
10 buffer by firmly tapping plate upside down on absorbent paper. Wipe of any
liquid from the bottom outside of the microtiter wells as any residue can
interfere in the reading step. All the washes should be performed similarly.
4. Add 100 µl of diluted Detection Antibody solution to each well, seal plate
and incubate at 37°C for 1 hour.
15 5. Wash plate 4 times with Wash Buffer (1X) as in step 3.
6. Add 100µl of diluted Streptavidin-HRP solution to each well, seal plate and
incubate at 37°C for 30 minutes.
7. Wash plate 4 times with Wash Buffer (1X) as in step 3. For this final wash,
soak wells in Wash buffer for 30 seconds to 1 minute for each wash. This
20 will help minimize background.
8. Add 100µl of TMB Substrate solution and incubate in the dark for 15
minutes 37°C. Positive wells should turn bluish in color. It is not necessary
to seal the plate during this step.
9. Stop reaction by adding 100µl of Stop Solution to each well. Positive wells
25 should turn from blue to yellow.
10. Read the absorbance at 450 nm within 30 minutes of stopping reaction.
37
Results:
In this study, Interleukin 10 (IL-10) levels were elevated from 34.83 ± 0.66 pg/ml
in the normal group to 57.79 ± 1.16 pg/ml in the HCOV disease control group.
The results of this study demonstrated reduction in IL-10 levels (P <0.001) in the
treatment group. The group in which the infected 5 mice were treated with a 150
µg/mice (low dose) and 300 µg/mice (high dose) of a Naringin test sample
showed reduction of 38.15 ± 0.17 pg/ml (with low dose) and 34.61 ± 0.8 pg/ml
(with high dose) in serum IL-6 levels compared to the disease control group.
? Mouse TNF-a ELISA: Quantification of TNF-a
10 I. Reagent Preparation
1. Wash Buffer (20X):
Dilution: To make wash buffer (1X), add 5ml of wash buffer (20X) to 95 ml
of DI water. This is the working solution.
2. Assay Diluent (5X):
15 Dilution: To make assay diluent (1X), add 10 ml of assay diluent (5X) to 40
ml of DI water. This is the working solution.
3. Biotin Conjugated Detection Antibody (100µl):
Dilution: Add 50 µl of detection antibody to 5950µl of assay diluent (1X) to
make final volume to 6 ml.
20 4. Concentrated Streptavidin-HRP (240 µl):
Diluent: Add 83.33 µl of concentrated streptavidin-HRP to 4916.67 µl of
assay diluent (1X) to make final volume to 5ml.
5. Standard (Mouse TNF-a; Lyophilized):
Reconstitution: Reconstitute lyophilized Mouse TNF-a standard with 50 µl
25 of Distilled water to achieve final concentration 0.5 µg/ml. Mix gently and
38
leave to stand for 15 mins before further dilution. Use immediately and do
not store reconstituted standard.
II. Assay Procedure
1. Bring all reagents to room temperature prior to use. A standard curve is
5 required for each assay.
2. Add 100 µl/well of Standards and Samples to the plate. Perform six twofold
serial dilutions of the 1000 pg/ml top standard, either within the plate or
in separate tubes. Thus, the Mouse TNF-a standard concentrations are 1000
pg/ml, 450 pg/ml, 225 pg/ml, 112.5 pg/ml, 56.25 pg/ml, 28.13 pg/ml, 14.06
10 pg/ml 7.03 pg/ml, 3.52 pg/ml and 0 pg/ml. Assay diluent (1X) serves as the
zero standard (0 pg/ml). Seal plate and incubate at 37°C for 2 hours.
3. Aspirate and wash plate 4 times with Wash Buffer (1X) and blot residual
buffer by firmly tapping plate upside down on absorbent paper. Wipe of any
liquid from the bottom outside of the microtiter wells as any residue can
15 interfere in the reading step. All the washes are to be performed similarly.
4. Add 100 µl of diluted Detection Antibody solution to each well, seal plate
and incubate at 37°C for 1 hour.
5. Wash plate 4 times with Wash Buffer (1X) as in step 3.
6. Add 100µl of diluted Streptavidin-HRP solution to each well, seal plate and
20 incubate at 37°C for 30 minutes.
7. Wash plate 4 times with Wash Buffer (1X) as in step 3. For this final wash,
soak wells in Wash buffer for 30 seconds to 1 minute for each wash. This
will help minimize the background.
8. Add 100 µl of TMB (3,3',5,5'-Tetramethylbenzidine) Substrate solution and
25 incubate in the dark for 15 minutes 37°C. Positive wells should turn bluish
in color. It is not necessary to seal the plate during this step.

ABSTRACT
5
ORAL COMPOSITION CONTAINING NARINGIN AND USES THEREOF
The present invention relates to an oral composition comprising an effective amount of
naringin, Zingiber officinale extract, and one or more other actives along with
10 pharmaceutically acceptable excipients; and a process for its preparation.
To,
15 The Controller of Patents
The Patent Office
At Mumbai ,CLAIMS:WE CLAIM:
1 . An oral composition comprising:
(a) naringin in an effective amount;
(b) Zingiber officinale extract in an effective amount;
(c) Vitamin C in an effective amount;
(d) zinc ion source in an effective amount;
(e) honey in an effective amount;
and one or more pharmaceutically acceptable excipients.
2. The oral composition as claimed in claim 1, wherein the composition comprises:
(a) 20 mg to 40 mg naringin;
(b) 8 mg to 16 mg Zingiber officinale extract;
(c) 12 mg to 25 mg Vitamin C;
(d) 2 mg to 5 mg zinc ion source;
(e) 80 mg to 160 mg honey;
and one or more pharmaceutically acceptable excipients.
3. The oral composition as claimed in claim 1, wherein the zinc source is selected from
zinc gluconate, zinc sulfate, or zinc acetate.
4. The oral composition as claimed in claim 1, wherein the composition is in the form of
lozenges, dragees, chewable tablets, chewing gums or capsules.
5. The oral composition as claimed in claim 1 or claim 4, wherein the composition is in
the form of a lozenge.
6. The oral composition as claimed in claim 5, wherein the lozenge comprises:
(a) 20 mg to 40 mg naringin;
(b) 8 mg to 16 mg Zingiber officinale extract;
(c) 12 mg to 25 mg Vitamin C;
(d) 2 mg to 5 mg zinc gluconate;
(e) 80 mg to 160 mg honey.
and one or more pharmaceutically acceptable excipients.
7. The oral composition as claimed in claim 5, wherein the lozenge comprises:
(a) 25 mg naringin;
(b) 10 mg Zingiber officinale extract;
(c) 15 mg Vitamin C;
(d) 3 mg zinc gluconate;
(e) 100 mg honey;
and one or more pharmaceutically acceptable excipients.
8. The oral composition as claimed in claim I or claim 4, wherein the composition is in the
form of capsules.
9. The oral composition as claimed in claim 8, wherein the capsules are selected from soft
capsules or hard capsules.
10. A process for the preparation of lozenges as claimed in claim 5 comprising:
(i) mixing sugar and water, and bringing it to boil to dissolve the mixture to obtain
a viscous mass;
(ii) heating the above viscous mass at 140°C, and passing it through vapo separation
chamber;
(iii) adding the actives such as naringin, Zingiber officinale extract; Vitamin C; zinc
ion source; honey to the mixture obtained in step (ii) above; and immediately
starting drawing ribbon with cooling;
(iv) adding flavouring agents during ribboning and cooling; and
(v) rolling, molding as lozenges, and drying.
Dated this 141h day of April 2022
To,
The Controller of Patents
The Patent Office
At Mumbai
JAYSHANKAR MENON
COMPANY SECRETARY & AVP-LEGAL
INDOCO REMEDIES LIMITED
(Applicant)