DESC:HERBO-MINERAL PREPARATION / MEDICINE TO IMPROVE IMMUNITY

Field Of The Invention
The invention relates to from 1 – 4 compositions preferably from 2 – 3 compositions containing from 5 – 10 rasayana herbal medicines and from 1 – 4 minerals in reduced dosage to impart or boost immunity to mammals.
Preferably, the invention relates to from 1 – 4 herbo-mineral compositions preferably from 2 – 3 compositions containing from 3 – 8 rasayana herbal medicines and from 1 – 4 minerals in reduced dosage to impart or boost immunity to mammals and particularly in boosting innate immunity of patients with COVID-19 infection.
Object of the invention
The object of the invention is to provide from 1 – 4 compositions preferably from 2 – 3 compositions containing from 5 – 10 rasayana herbal medicines and from 1 – 4 minerals in reduced dosage to impart or boost immunity to mammals.
Preferably, the invention relates to from 1 – 4 herbo-mineral compositions preferably from 2 – 3 compositions containing from 3 – 8 rasayana herbal medicines and from 1 – 4 minerals in reduced dosage.
The rasayana herbal medicines are preferably in the form of ‘Ghan’ and minerals are preferably in the form of ‘Bhasma’ or in ‘Colloidal’ or in ‘Nano particulate’ form.
Another object is to provide at least 2 – 3 compositions containing from 5 – 10 rasayana herbs and from 1 – 4 minerals in such a way that one composition is a starter composition and second (and optionally additionally third) composition is / are continuation composition / compositions.
Summary of the invention
Under the first aspect, invention provides around 5 – 10 rasayana herbs in the form of powders / Ghan with 1 - 4 minerals preferably in the form of bhasma or in colloidal or in nano particulate form in such a way that they can be combined in two or more preparations / compositions and administered together, simultaneously or sequentially to impart / boost immunity.
Second aspect is to provide at least 2 – 3 compositions containing from 5 – 10 rasayana herbs and from 1 – 4 minerals in such a way that one composition is a starter composition and second (and optionally additionally third) composition is / are continuation composition / compositions.
Preferably, the invention relates to from 1 – 4 herbo-mineral compositions preferably from 2 – 3 compositions containing from 3 – 8 rasayana herbal medicines and from 1 – 4 minerals in reduced dosage.
Reduced dosage means dose is reduced to 1 – 50 % of the usual dose of herbal medicine or mineral. Preferably, dose is reduced to 1 – 25 % of the usual dose of herbal medicine or mineral.
Composition can be tablet, capsule, caplets, coated tablets, multi-layered tablets, minitablets, pill, powder, granules, seeds, pellet, extrudates, spheronized material, compacts, particulate preparation, dispersion, dispersible tablet, fast disintegrating tablet, lozenge, sublingual or buccal tablets, powder mixture, syrup, suspension, powder for oral solution, powder for oral suspension injectables, liquid, solution, subcutaneous, intravenous, intramuscular composition.
The total content of at least 3 – 8 herbal medicines and 1 – 4 minerals in a single composition is from 200 mg – 800 mg, preferably from 250 mg to 700 mg, more preferably from 300 – 600 mg and most preferably from 315 mg to 575 mg.
Brief Description of Drawings
Figures 1 – 3 provide preparation of Ghan by aqueous / water extract or by super critical extraction.
Figure 4 provides number of patients screened, enrolled and given drug i.e. Cap IP (IP1 + IP 2) or placebo in the present study.
Figure 5 provides number of patients enrolled for the study over time and gender distribution of patients enrolled. Average age of enrolled patients = 46 years.
Figure 6 provides a flow chart indicating steps from screening, enrollment and first, second and third follow up after administration of Cap IP or placebo.
Figure 7 provides antibody concentrations of IgG and IgM in serum RU/ml over period after onset of symptoms.

Background Of The Invention
Recent time is marked with a Coronavirus pandemic which is a threat to entire humanity. In absence of any vaccine, and medication that can effectively tackle all mutants and all stages of the virus, the larger emphasis is placed on immunity of the individual.
Older population having low immunity is at higher risk than ever before.
India’s older population (age 60 and above) is projected to climb from 8 % in 2010 to 19 % in 2050. This population should be disease and disability free with high physical and cognitive function and active engagement with life. This is possible with enhancement in immunity.
Immunity may be defined as the body’s ability to identify and resist large numbers of infectious and potentially harmful microorganisms, enabling the body to prevent or resist diseases and inhibit organ and tissue damage. Immunity also plays a vital role in maintaining health and prolongs the ageing process. It also plays important role in fighting dreadful diseases like cancer etc.
The immune system is not confined to any one part of the body. The immune system is made up of special organs, cells and chemicals that fight infection (virus, bacteria, fungi etc.). The main parts of the immune system are: white blood cells, antibodies, the complement system, the lymphatic system, the spleen, the thymus, and the bone marrow. These are the parts of your immune system that actively fight infection.
The immune system keeps a record of every microbe it has ever defeated, in types of white blood cells (B- and T-lymphocytes) known as memory cells. This means it can recognise and destroy the microbe quickly if it enters the body again, before it can multiply and make you feel sick.
Some infections, like the flu and the common cold, have to be fought many times because so many different viruses or strains of the same type of virus can cause these illnesses. Strong Immunity is the Defense to Protect from many infections including Viral infection (e.g.Corona).
Immune stem cells, formed in the bone marrow, may remain in the bone marrow until maturation or migrate to different body sites for maturation. Subsequently, most immune cells circulate throughout the body, exerting specific effects. The immune system has two distinct but overlapping mechanisms with which to fight invading organisms, the antibody-mediated defense system (humoral immunity) and the cell-mediated defense system (cellular immunity).
Nature has been an excellent source of therapeutic agents since the time immemorial. Traditional and conventional medicines are mostly based on numerous natural resources, including plants which are being used as a primary source in folkloric medicine. Before the discovery of chemical and synthetic compounds, these plants played a significant role as a traditional medicine for curing various diseases throughout the world. However, with the emergence of modern techniques and equipment, synthesis of new classes of synthetic compounds has attracted attention among researchers during recent years. Some of these compounds were first isolated from the natural sources but further modified to improve their efficacy

Detailed description of the invention
The words preparation and compositions are used synonymously.
The composition can be selected from tablet, capsule, caplets, coated tablets, multi-layered tablets, minitablets, pill, powder, granules, seeds, pellet, extrudates, spheronized material, compacts, particulate preparation, dispersion, dispersible tablet, fast disintegrating tablet, lozenge, sublingual or buccal tablets, powder mixture, syrup, suspension, powder for oral solution, powder for oral suspension injectables, liquid, solution, subcutaneous, intravenous, intramuscular composition.
In an embodiment, the composition is a capsule. In another embodiment, the composition is tablet. In one more embodiment, the composition is syrup or suspension. In yet another embodiment, the composition is powder, powder for oral suspension, powder for oral solution or granules.
Multi-layered tablets, coated tablets and modified release compositions are also prepared where release is modified to suit sustained release, extended release, delayed release of herbal medicines and minerals. Alternatively, one or few herbal medicine or mineral can be in modified release form and other herbal medicines and minerals are in immediate release form.
Composition can be tablet, capsule, caplets, coated tablets, multi-layered tablets, minitablets, pill, powder, granules, seeds, pellet, extrudates, spheronized material, compacts, particulate preparation, dispersion, dispersible tablet, fast disintegrating tablet, lozenge, sublingual or buccal tablets, powder mixture, syrup, suspension, powder for oral solution, powder for oral suspension injectables, liquid, solution, subcutaneous, intravenous, intramuscular composition.
The compositions of the present invention preferably include tablet, dragee, capsule, caplet, orally-disintegrating tablet, film-coated tablet, enteric tablet, buccal tablet, sublingual tablet, chewable tablet, effervescent tablet, slow-release tablet, rapid-release tablet, modified-release tablet, delayed-release tablet, prolonged- release tablet, controlled-release tablet, sachet, granule, pilule, powder, pellet, , pastille and similar solid oral dosage forms.
Other excipients are chosen based on the composition to facilitate the physical formulation of various dosage forms. Pharmaceutical composition of the present inventions may comprise along with the active and a solubilizer, other pharmaceutically acceptable excipients like diluents / fillers, disintegrants, binders, lubricants, glidants, and the mixtures thereof, to facilitate the physical formulation of various dosage forms.
Binders can be selected from the group, but are not limited to methylcellulose, sodium carboxymethylcellulose, calcium carboxymethylcellulose, ethyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, silicified microcrystalline cellulose(SMCC), polyvinyl pyrrolidone, gelatine, polyvinyl alcohol, acacia, tragacanth, guar, pectin, starch paste, pregelatinized starch, alginic acid, compressible sugar, liquid glucose, dextrates, dextrin, dextrose, maltodextrin, guar gum, magnesium aluminium silicate, polymethacrylates, sorbitol and other materials known to one of ordinary skill in the art. A mixture of binders may also be used. The binder is preferably used in an amount of from about 2 to about 15% by weight.
Disintegrant can be selected from the group, but are not limited to alginic acid, carboxymethylcellulose calcium, carboxymethyl cellulose, carboxymethylcellulose sodium, cross-linked sodium carboxymethylcellulose, low substituted hydroxypropyl cellulose, colloidal silicon dioxide, croscarmellose sodium (CCS), crospovidone, guar gum, magnesium aluminium silicate, microcrystalline cellulose (MCC), methyl cellulose, polyvinylpyrrolidone, cross-linked polyvinylpyrrolidones, polacrifin potassium, starch, pregelatinized starch, sodium alginate, sodium lauryl sulphate, sodium starch glycolate, crystalline cellulose, hydroxypropyl starch and other materials known to one of ordinary skill in the art. The combination of above-mentioned disintegrants can also be used.
Preferably, the disintegrant is selected from the group consisting of croscarmellose sodium, crospovidone and sodium starch glycolate. Disintegrant is preferably used in an amount of from about 1% to about 20% by weight of the tablet, more preferably in an amount of from about 2% to about 15% by weight and most preferably in an amount of from about 2% to about 10%.
In an embodiment, disintegrant is 3.2 % of the core tablet weight. In another embodiment, disintegrant is 4.6 % of the core tablet weight. In few embodiments, the disintegrant is from 5.5 to 5.7 % by weight of the tablet.
Diluents or filler can be selected from the group consisting of calcium carbonate, calcium phosphate, calcium sulphate, carboxymethylcellulose calcium, carboxymethylcellulose sodium, compressible sugar, confectioner's sugar, dextrates, dextrin, dextrose, dibasic calcium phosphate dihydrate, dibasic calcium phosphate, fructose, lactose, maize starch, magnesium carbonate, magnesium oxide, maltodextrin, mannitol, maltitol, microcrystalline cellulose, powdered cellulose, pregelatinized starch, sorbitol, starch, sucrose, sugar spheres, talc, tribasic calcium phosphate, and xylitol and other materials known to one of ordinary skill in the art.
Preferred diluents or fillers include lactose, fructose, starch, sucrose, mannitol, maltitol, sorbitol, xylitol and microcrystalline cellulose.
Binder can be selected from the group, but is not limited to methylcellulose, sodium carboxymethylcellulose, calcium carboxymethylcellulose, ethyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, silicified microcrystalline cellulose(SMCC), polyvinyl pyrrolidone, gelatine, polyvinyl alcohol, acacia, tragacanth, guar, pectin, starch paste, pregelatinized starch, alginic acid, compressible sugar, liquid glucose, dextrates, dextrin, dextrose, maltodextrin, guar gum, magnesium aluminium silicate, polymethacrylates, sorbitol and other materials known to one of ordinary skill in the art. A mixture of binders may also be used. The binder is preferably used in an amount of from about 1 to about 15% by weight, preferably from about 1 – 10 % by weight of the tablet.
Lubricants can be selected from the group consisting of vegetable oils, such as hydrogenated vegetable oil or hydrogenated castor oil; polyethylene glycols, such as polyethylene glycol (PEG)-4000 and PEG-6000; stearic acid; derivatives of stearic acid, such as magnesium stearate, sodium stearate, calcium stearate, zinc stearate, glyceryl monostearate, glyceryl palmitostearate and sodium stearyl fumarate; mineral salts, such as talc; inorganic salts; organic salts, such as sodium benzoate, sodium acetate, sodium chloride and sodium oleate; and polyvinyl alcohols, microcrystalline cellulose, sodium lauryl sulphate, silica, colloidal silica, corn-starch, calcium silicate, magnesium silicate, silicon hydrogel and other materials known to one of ordinary skill in the art.
The preferred lubricant is magnesium stearate. Preferably, lubricant is used in an amount from about 1 to about 5% by weight.
Glidants can be selected from the group, but is not limited to colloidal silicon dioxide, colloidal silica, corn-starch, talc, calcium silicate, magnesium silicate, magnesium trisilicate, amorphous silica, colloidal silicon, silicon hydrogel, powdered cellulose, silicon dioxide, talc, and other materials known to one of ordinary skill in the art.
Preferably, talc is used as a glidant. More preferably, colloidal silicon dioxide is used as a glidant. Most preferably, combination of talc and colloidal silicon dioxide is used as a glidant.
Glidants are used in an amount from about 0.5 to about 10 % by weight of the core tablet. Preferably, from about 1 to about 5 percent by weight of the tablet.
Traditional medical practitioners use botanicals and other natural substances to stimulate or potentiate the body's own defence mechanisms. This way the body's natural balance is preserved, and side effects are either not present or substantially reduced. Although both vaccines and botanicals elicit an immune response, vaccines boost the body's response to a specific pathogen whereas botanicals tend to enhance overall immunity. Immune-potentiating botanicals are also used to increase the effectiveness of drugs and reduce side effects.
Keeping population healthy and boosting with immunity is challenging and presently a lot of research has been undertaken for achieving this goal.
In Ayurveda, Rasayana is a specialized therapy practicised since ages by the ancient Indian physician to combat the adverse effect of senility and keep the
youngness prolonged. It is the Ayurvedic practice of rejuvenation therapy. Rasayana is the preventive therapy which is helpful to maintain health, retard aging process and promote immune system to fight against infection.
The common medicines in Ayurveda with Rasayana properties include
Emblica officinalis, Sida cordifolia, Semicarpus, anacardium, Tinospora cordifolia, Centella asiatica, Piper longum, Zingiber officinale Convolvulus pluricaulis, Asparagus racemosus, Anethumsowa, Shilajit, Terminalia chebula , Embelia ribes, Glycyrrhiza glabra and Withania somnifera.
The present inventors have come up with crucial combination of rasayana herbs with minerals in specific doses such that at least three and up to eight rasayana herbs and from 1 – 4 minerals are administered to boost immunity. The administration comprises administering from 1 – 4 compositions, preferably up to 2 – 3 compositions containing from 5 – 10 rasayana herbs and from 1 – 4 minerals.
The total content of at least 3 – 8 herbal medicines and 1 – 4 minerals in a single composition is from 200 mg – 800 mg, preferably from 250 mg to 700 mg, more preferably from 300 – 600 mg and most preferably from 315 mg to 575 mg.
Particularly, the present invention relates to from 1 – 4 herbo-mineral compositions preferably from 2 – 3 compositions containing from 3 – 8 rasayana herbal medicines and from 1 – 4 minerals in reduced dosage to impart or boost immunity to mammals and particularly in boosting innate immunity of patients with COVID-19 infection.
When two – four compositions are used together i.e. together, simultaneously, on same day or in the same month or sequentially one after the other viz. after treatment of first finishes, treatment with second commences, or in any way when their administration or treatment provided by employing them is related in some way for prophylactic or therapeutic purpose, they are referred as a first herbomineral composition, a second herbomineral composition, a third herbomineral composition or a fourth herbomineral composition.
When the compositions are sent for any human trials they are referred as Investigational products.
In various embodiments a first herbomineral composition and a second herbomineral composition is prepared. In another embodiment, a first, a second and a third herbomineral compositions are prepared.
From the group of compositions, a first herbomineral composition containing three herbal medicines and two minerals and second herbomineral composition containing six herbal medicines and 1 mineral are chosen for various human trials.
Herbs are preferably in the form of Ghan and Minerals are preferably in the form of bhasma or in colloidal or in nano particulate form. Any suitable method can be used to prepare Ghan. Alternatively, minerals can be in their oxides, hydrates, solvates and salt forms. For example, if calcium is chosen as a mineral, it can be provided in inorganic or organic form such as calcium chloride, calcium carbonate, calcium phosphate, tricalcium citrate, calcium lactate, calcium lactate gluconate, calcium gluconate and calcium orotate.
Preferred zinc form is calcinated zinc, zinc oxide or salts with acetate, gluconate, and sulfate.
Various human trials are conducted using the compositions of the present invention including a pilot study and a study in Covid-19 positive patients.
Pilot study
A Pilot study is conducted as follows:
A study was done in group of 100 patients –
All patients required immunity medicine. The patients comprised of those with various disorders including cancer with advanced stage either undergone major surgery and / or on chemotherapy, chronic kidney disease, general debility, recurrent infections, aged population etc. Age group is from 18 to 80 yrs.
5 groups each of 20 was made such as groups A,B,C,D,E. Various compositions / herbal rasayana optionally with one or more minerals having immunomodulatory properties like anti oxidative, anti cancer, anti viral ,immune boosting ,anti degenerative etc. were selected. All groups were given different compositions containing increasing number of rasayana herbs as provided in table 1A. It was observed that more the number of rasayana herbs, better clinical results were obtained . Another group was given all 9 rasayana herbs along with minerals (herbo-mineral) was given with idea that minerals will further potentiate the action on plant extracts, will improve the cellular metabolism, will accelerate the action .Group E (herbo-mineral) showed significantly better results as compared to all other groups.
Following compositions are administered to above groups:
Group A – Composition 1
Group B – Composition 2
Group C – Composition 3
Group D – Composition 4
Group E – Composition 5
Each herbal rasayana medicine can be used in amount up to 250 mg.
Following table provides various Compositions 1 – 4 in which the rasayana herbs and minerals are combined.
Table 1A: Compositions of pilot study
Dose Composition 1 Composition 2 Composition 3 Composition 4 Composition 5A
25 – 250 mg, preferably 25 – 100 mg Zingiber officinaleghan Zingiber officinaleghan Zingiber officinaleghan Zingiber officinaleghan Zingiber officinaleghan
25 – 250 mg, preferably 25 – 150 mg Embelia ribes ghan Embeliaribesghan Embeliaribesghan Embelia ribes ghan Embelia ribes ghan
25 – 250 mg, preferably 25 – 100 mg Glycyrrhiza glabra ghan Glycyrrhiza glabra ghan Glycyrrhiza glabra ghan Glycyrrhiza glabra ghan Glycyrrhiza glabra ghan
25 – 250 mg, preferably 25 – 150 mg Terminalia chebulaghan Terminalia chebulaghan Terminalia chebulaghan Terminalia chebulaghan Terminalia chebulaghan
25 – 250 mg, preferably 25 – 150 mg Tinosporacordifoliaghan Tinosporacordifoliaghan Tinosporacordifoliaghan Tinosporacordifoliaghan Tinosporacordifoliaghan
25 – 250 mg, preferably 25 – 100 mg Asparagus racemosus ghan Asparagus racemosus ghan Asparagus racemosus ghan Asparagus racemosus ghan
25 – 250 mg, preferably 25 – 100 mg Emblica officinalis ghan Emblica officinalis ghan Emblica officinalis ghan
Up to 50 mg preferably up to 10 mg Piper longumghan Piper longumghan
25 – 250 mg, preferably 25 – 150 mg Withania somniferaghan Withania somniferaghan
Up to 250 mg, preferably from 5 mg to up to 150 mg shankhbhasma
Up to 250 mg, preferably from 5 mg to up to 100 mg jasathbhasma

The composition 5A may additionally contain Convolvulus pluricaulis from 25 mg – 150 mg (5B composition).
Interestingly, second part of the study was to check if the compositions containing from 5 – 10 rasayana herbs and from 1 – 4 minerals can be given in the form of 2 to 3 compositions such that one composition is a starter composition and second (and / third composition) is (are ) continuation compositions as provided in table 1B. Accordingly, from composition 5A containing 9 rasayana herbs and two minerals, different starter compositions and continuation compositions are prepared. In this, 9 rasayana herbs are divided into 3 compositions each containing 3 rasayana herbs and each composition contained at least one mineral . After few trials, the starter composition and continuation composition as provided in table B are prepared.

Table 1B – Starter composition and Continuation composition.
A small study is being planned after initial assessment which has surprisingly shown that composition 7A amd 7B are very promising in boosting immunity in all patients.

Dosage Composition 6A
Starter composition
IP 1 Composition 6B
Continuation composition
IP 2 Dosage Composition 7A
Starter composition Composition 7B
Continuation composition
25 – 250 mg, preferably 25 – 100 mg Zingiber officinaleghan 60 mg Zingiber officinaleghan
25 – 250 mg, preferably 25 – 150 mg Embeliaribesghan 105 mg Embeliaribesghan
25 – 250 mg, preferably 25 – 100 mg Glycyrrhiza glabra ghan 60 mg Glycyrrhiza glabra ghan
25 – 250 mg, preferably 25 – 150 mg Terminalia chebulaghan 102 mg Terminalia chebulaghan
25 – 250 mg, preferably 25 – 150 mg Tinosporacordifoliaghan 75 mg Tinosporacordifoliaghan
25 – 250 mg, preferably 25 – 100 mg Asparagus racemosus ghan 60 mg Asparagus racemosus ghan
25 – 250 mg, preferably 25 – 100 mg Emblica officinalis ghan 65 mg Emblica officinalis ghan
Up to 50 mg preferably up to 10 mg Piper longumghan 2 mg Piper longumghan
25 – 250 mg, preferably 25 – 150 mg withaniasomniferaghan 109 mg withaniasomniferaghan
Up to 250 mg, preferably up to 150 mg shankhbhasma 125 mg shankhbhasma
Up to 250 mg, preferably up to 100 mg jasathbhasma jasathbhasma 20 mg jasathbhasma jasathbhasma

Particularly, it has been surprisingly found that 3 – 8 herbal medicines and 1-4 minerals combined in two different compositions are efficacious in boosting innate immunity of patients with COVID-19 infection.
The total content of 3 herbal medicines and 2 minerals in a single composition was about 400 mg. The total content of 6 herbal medicines and 1 mineral in a single composition was about 450 mg.
These compositions are tried as starter and continuous compositions where one is given in a starter treatment (start of the treatment) for a few days and the other is given in a continuous treatment.
These compositions are also given in a fixed treatment, each for 5 – 15 days both together and sequentially.
In one more embodiment, the treatment is given in such a way that both first and second herbomineral compositions are given together for a few days from 5 – 15 days and one of the first or second composition is given for an additional equal duration. This has been found very effective symptomatically.
In further trials instead of 1 dosage form, two dosage forms are given everyday.
Study in Covid-19 positive patients
In yet another embodiment, the treatment is given in such a way that both first and second herbomineral compositions are given together twice a day for 15 days and the second composition is given twice a day for an additional equal duration of 15 days in Covid-19 positive patients. This trial has been described in details in the specification.
The inventors have invented compositions containing reduced dosage of some herbal medicines combined with 1-4 minerals. These compositions are IP 1 and IP 2 as provided in table 1C. These compositions are filled in the capsule and administered in the study. These compositions are subjected to a clinical trial which is a single centred, randomized, 2-arm, parallel group, double blind, 1:1 ratio, controlled, exploratory trial with a study period of 30 days from the day of enrolment.

Table 1C: Composition Sent for Clinical trials

Sr. No. Product Capsule IP 1
1 Zingiber officinale (Ginger) 60 – 120 mg
2 Embelia ribes (Vavding) 75 – 125 mg
3 Glycyrrhiza glabra (Licorice) 60 – 120 mg
4 Zinc (Jasad Bhasma) 20 – 60 mg
5 Calcium (Shankh Bhasma) 100 – 150 mg
Total content per unit dosage form 315 mg – 575 mg
Capsule IP 2
1 Terminalia chebula Haritaki 100 – 150 mg
2 Tinospora cordifolia Guduchi 60 – 120 mg
3 Asparagus racemosus Shatavari 40 – 80 mg
4 Emblica officinalis Amla 60 – 120 mg
5 Withania somnifera / Ashwagandha 100 – 150 mg
6 Long pepper 0.05 – 20 mg
7 Zinc Jasad Bhasma
8 Total content per unit dosage form 360.05 mg – 640 mg

Participants of the study were Patients attending the COVID treatment centre at Yashwantrao Chavan Memorial Hospital, Nehrunagar, Pimpri, Pune, India. These were screened for their participation in the study. Patients who were known COVID-19 positive(with positive RT-PCR), eligible and willing were enrolled in the study.
Intervention and comparator
Intervention Arm: Two capsules, Investigational Product (IP1) - 400mg and Investigational Product (IP2) 450mg, containing herbal extracts (a blend of water and CO2extracts) of Shunthi (Zingiber officinale(Ginger), Vidanga (Embelia ribes), Yashtimadhu (Glycyrrhiza glabra), Haritaki (Terminalia chebula), Guduchi (Tinospora cordifolia), Shatavari (Asparagus racemosus), Aamalaki (Emblica officinalis), Pippali (Piper longum) and calcined Zinc, Shankha bhasma.
Placebo Arm: Edible starch ~ 450 mg. The look and feel of IP and of Placebo boxes were very similar.
72 patients were enrolled with 37 into the intervention group and 35 into the placebo group
Patients took two capsules (one each of IP-1 and IP-2) twice a day for 15 days, and from the 16th day, one capsule of IP-2 twice a day up-to day 30. Capsules are administered orally with plain water.
The IP 1 and IP 2 are to be taken with all other concomitant medicines prescribed by the treating physician/doctor.
The dose of each component in the IP is very safe to administer. The investigational
Products are registered products with the Indian Government andhave been used for more than 6 months in various health conditions but not for COVID-19.
The outcome of the above treatment was as follows:
Primary outcome:
Efficacy of the herbal extracts in COVID 19 positive patients (in declining viral load: time-point: 4 days and early recovery).
Secondary Outcomes:
i) Efficacy of the herbal extractsas an immune-modulator - TH1, TH2, Th17, IL6, NK Cells and CD markers; Immunoglobulin IGG (Serum); Immunoglobulin IGM (Serum) at 30 days.
ii) Efficacy of the investigational product in reducing sequela of the disease
iii) Safety analysis (Liver Function Test and Kidney Function Test) including serious allergic reaction of: rash, itching/swelling, severe dizziness, trouble breathing.
Therefore, it can be concluded that herbommineral compositions of the present invention can be together and sequentially administered in Covid-19 treatment.
Various test data is presented in tabular form in the specification.
The advantages of the invention is as follows.
Usually, rasayana herbs are administered in high doses such as 500 mg to 2 g in one or 2 - 4 divided doses. Brahma Rasayana available for sale are usually in the form of Churna and daily dose is 10 g minimum. By combining from 5 – 10 rasayana herbs or 3 -8 herbal medicines and from 1 – 4 minerals in preferably 1 – 4 and most preferably 2 – 3 compositions, inventors are able to employ much lower doses than recommended to achieve same or even better effects and without any side effects.
Thus, it is possible to combine many rasayana medicines in just 1 – 4 compositions / preparations, preferably, 2-3 compositions / preparations which otherwise are separately administered because of their individual large doses and because it is cumbersome to take 5 – 10 different preparations / compositions.
Since the present composition provide ways to combine 5 – 10 different herbal rasayana medicines or 3 -8 herbal medicines with 1- 4 minerals, the synergistic action of all herbal and mineral preparations greatly reduced the total daily intake of each medicine. Ginger when used in solid form i.e. either dry powder / ghan form, the daily dose of 1 g - 1.5 g is reduced from 25 – 250 mg, and most preferably, 25 – 150 mg or 25 – 100 mg and even such reduced doses have been found sufficient and produce same or even better action.
Preferably the rasayana herbs selected are those which are reported to have immunomodulatory actions such as Emblica officinalis, Tinospora cordifolia, Piper longum, Zingiber officinale, Asparagus racemosus, Terminalia chebula , Embelia ribes, Glycyrrhiza glabra, Withania somnifera and Convolvulus pluricaulis.

Zingiber officinale
Ginger is a spice consumed worldwide for culinary and medicinal purposes.
Ginger plant has a number of chemicals responsible for its medicinal properties, such as antiarthritis, antiinflammatory, antidiabetic, antibacterial, antifungal, anticancer, etc.Ginger contains many volatile oils (sesquiterpenes) and aromatic ketones (gingerols). Gingerols are believed to be the more pharmacologically active constituents of ginger.
Nathorn Chaiyakunapruk et al has concluded that a fixed dose at least 1 g of ginger is more effective than placebo for the prevention of postoperative nausea and vomiting and postoperative vomiting.
Ginger is one of the most effective natural immunomodulator [Radha Singh*, and Kusum Singh, 2019]. An in vitro study found that ginger inhibited lymphocyte proliferation; this was mediated by reductions in IL-2 and IL-10 production [Wilasrusmee C et al, 2002]. Aqueous ginger extract significantly increased the production of IL-1ß, IL-6 and TNF-a inactivated peritoneal mouse macrophages and splenocyte proliferation and cytokine production [Ryu HS and Kim HS, 2004]. Ginger rhizome diet for 12 weeks showed increased haematocrit, haemoglobin, erythrocyte, MCH, MCHC, WBC values and neutrophils percentage. Ginger essential oil showed improvement in humoral and cell mediated immune response in immune suppressed mice. [Carrasco FR et al, 2009]. The powdered ginger rhizome is capable to improve non-specific immune response in rainbow trouts [Haghighi M, Rohani MS, 2013].
Thus, Zingiber officinale is selected one of the herbal medicine to prepare compositions of the present invention.
Andrea Gordon, and Abigail Love under chapter 54 of Integrative Medicine (Fourth Edition) 2018, Pages 542-549.e2 provide various doses used by various researchers for Zingiber officinale. These doses are 1000 mg daily in 2 -3 divided doses or a higher dose of 650 mg three times daily. It is further mentioned that the total dose of less than 1500 mg daily is more effective.

2.Embelia ribes-
Embeliaribes comes under rasayana plants. (Rasayana implies to the circulation of nutrients). Rasayana is a substance that nourishes and tones the entire body.
Gupta et al (Asian Jr. Multidisciplinary Studies, 2015, Vol.3(2))
cites reference of Bhandari U et al (2007) providing that the ethanolic extract of Embeliaribes enhances the antioxidant defense against reactive oxygen species produced under hyperglycemic condition and this protects ß-cells against loss, and
exhibit antidiabetic property. Embeliaribes extract has shown to enhance antioxidant defense and neuroprotective activity.
Wang P et al (2020) has reported that their findings indicate that the use of an adjuvant of embelin, a small molecular inhibitor of XIAP, increased the replication of oncolytic vaccinia virus (OVV) by mitigating antiviral innate immunity and further indicate utility of embelin as an adjuvant for oncolytic viro-immunotherapy.

3.Terminalia chebula-
This herb is extremely rich in medicinal properties. It is a constituents of the popular Ayurvedic formula Triphala (which contains equal proportions of Haritaki, Bahera (Terminalia bellerica) and Amla (Emblica officinalis).
It directly acts on the gastro-intestinal tract and reduces the passage of harmful toxins to the liver and kidneys.
Terminalia chebula contains several antiaging phenolic compounds are present in T. chebula. It includes gallic acid, chebulinic acid, isoterchebulin, punicalagin and 1,3,6-tri-O-galloyl-d-glucopyranosehave. These phenolic compounds have antioxidant activities.
H. N. Shivprasad et al’s study (2006) has shown that Terminalia chebula extract displays a dose-dependent immunostimulatory effect in relation to antigenic s Terminalia chebula extract produced a dose-dependent increase in both the parameters (i.e., antibody production and delayed-type hypersensitivity). Therefore, they concluded that the aqueous extract of fruit of Terminalia chebula has promising immunostimulant properties.
Belapurkar, Pranoti et al (2014) reports work of several researchers wherein T. chebula has been reported to be an effective antibacterial agent against a wide range of Gram-positive and Gram-negative bacteria, antifungal agent against various pathogenic fungi and an antiviral agent against swine influenza A virus, HSV-1, HIV-1 and cytomegalovirus.
Further the same paper reports studies conducted by various researchers
i) that have also inferred that the fruits of T. chebula act strongly against HBS antigen, inhibit HBV DNA polymerase and also significantly increased IFN-? and IL-2 levels in peripheral blood mononuclear cell culture, thus emphasizing its antiHBVactivity,;
ii) that have indicated it to be used as an antioxidantantiinflammatory, antianaphylactic, antimutagenic, antinociceptive and in wound healing activities;
iii) that indicated that the biologically active compounds such as chebulagic acid, gallic acid and ellagic acid make T. chebula highly potent antioxidant, which may be responsible for its immunomodulatory activity; and
iv) that indicates that its extract neutralizes reactive oxygen species (ROS) and scavenges free radicals. The free radicals are responsible for causing inflammation by stimulating release of cytokines such as IL-1, TNF-a and IFN-ß, which stimulate additional neutrophils and macrophages at site of inflammation.
Belapurkar et al (2014) further mentions that different antioxidants of the T. chebulaextract exhibit immunosuppressive properties, which help in neutralizing these important inflammatory mediators.

4.Tinospora cordifolia-
Tinosporacordifolia is an important medicinal plant. Through centuries, it has been extensively used in traditional medicinal reparations for treating various ailments. A variety of plant-derived materials polysaccharides, lectins, peptides etc. have been reported to stimulate immune system. Extract of T. cordifoliaposses good immunomodulatory activity.
Aher and Wahi (2010) reported that an oral administration of T. cordifolia alcoholic extract (100 mg/kg, p. o) in male wister rats was found to increase foot pad thickness which indicates immunomodulatory effects of T. cordifolia as compared to vehicle and cyclophosphamide treated groups.
5.Asparagus racemosus-
Asparagus racemosus is an important medicinal plant of tropical and subtropical India. Its medicinal use has been accounted in Indian and British pharmacopoeias and in indigenoussystem of medicine.
The dried roots of the plant are used as drug. The roots are said to be tonic and diuretic and galactgogue, the drug has ulcer healing effect probably via strenthening the mucosal resistance or cytoprotection. It has also been identified as one of the drugs to control the symotoms of AIDS. A. racemosus has also been successfully by some Ayurvedic practitioner for nervous disorder, inflammation and certain infectious disease (Shashi Alok et al, 2013)
Sharma M. et al (2016) cites paper by Gautam et al (2009) mentioning that Asparagus racemosus extract has shown immunoadjuvant potential and another paper by Goyal, Singh et al (2003) mentioning oral administration of decoction of powdered root of A. racemosus has been reported to produce leucocytosis and predominant neutrophilia along with enhanced phagocytic activity of the macrophages and polymorphs.
Pooja Shaha and Anurag Bellankimath report as follows:
i) Racemosus root extract modulated action of immune system;
ii) It induces the immune system to fight against immune deficiencies and infections;
iii) It might be helpful in obtaining higher protective antibody against different vaccinations including more effective cell mediated immune response for protection against various bacterial, viral and other diseases.
Akansha Singh & B. Sinha (2014) report that Several workers have studied the effect of A. racemosus root extract in augmentation of humoral and cell mediated immune responses providing better protection level against infections.

6.Emblica officinalis-
Emblica officinalis enjoys a hallowed position in Ayurveda-an Indian indigenous system of Medicine. According to believe in ancient Indian mythology, it is the 1st tree to be created in the universe (Grover, Herpreet Singh et al, 2020).
Fruit and its extract is widely used as phytomedicines. It is one of the richest source of natural vitamin C and low molecular weight hydrolysable tannins. This makes it a good source of antioxidant. It has antimicrobial activities which includes antibacterial, antifungal and antiviral properties.
Charmkar N.K and Singh, Rajesh report beneficial role of Emblica officinalis in cancer, diabetes, liver treatment, heart trouble, ulcer, anemia and various other diseases and particularly its application as antioxidant, immunomodulatory, antipyretic, analgesic, cytoprotective, antitussive and gastroprotective. Further they particularly report role of Emblica officinalis to modulate the immune system and the inflammatory response.
Suja RS et al evaluated aqueous extract of dried Emblica officinalis Gaertn. (Amla) fruit pulp powder for immunomodulatory effect on male Swiss Albino mice. The mice were divided into three groups. The first group received vehicle alone to serve as control. The second and third groups received the extract orally at 100 and 200 mg/kg body weight dose levels respectively per day for a period of 19 days. Suja RS et al further reports that there was significant dose dependent increase in haemagglutination antibody titre, sheep red blood cells induced delayed type of hypersensitivity reaction, macrophage migration index, respiratory burst activity of the peritoneal macrophages, total leukocyte count, percentage lymphocyte distribution, serum globulin and relative lymphoid organ weight in Emblica treated mice indicating its ability to stimulate humoral as well as cell mediated immunity.

7.Withania somnifera-
Ziauddin, M et al report that W. somnifera prevented myelosuppression in mice treated with immunosuppressive drugs . It was observed in Ashwagandha-treated mice as compared with untreated (control) mice. We also report an immunostimulatory activity treatment with W. somnifera was accompanied by significant increases in hemolytic antibody responses towards human erythrocytes.
L. Davis et al (2002) mention that the role of W. somnifera as immunomodulator has been extensively studied. In a mouse study, WS root extract enhanced total white blood cell count. In addition, this extract inhibited delayed-type hypersensitivity reactions and enhanced phagocytic activity of macrophages when compared to a control group.
L. Davis and G. Kuttan, (2000) report that administration of W. somnifera also showed an enhancement in phagocytic activity of peritoneal macrophages when compared to control in mice. These results confirm the immunomodulatory activity of W. somnifera extract in indigenous medicine.

8.Piper longum -
Chauhan Khushbu et al (2011) report that tests such as haemagglutination titre (HA), macrophage migration index (MMI) and phagocytic index (PI) have demonstrated the immunostimulatory action of P.longum to be both specific and non specific.
Piper longum is well known for its medicinal and pharmaceutical importance.
Tripathi D. M et al report that the specific and nonspecific immune-stimulatory actions of P. longum fruits have been evaluated by hemagglutination titer, macrophage migration index, and phagocytic index. It is found to activate macrophages, as shown by an increased macrophage migration index and phagocytic index, indicating immune-stimulatory activity.

9. Glycyrrhizra glabra-
The name glycyrrhiza is derived from Greek words meaning "sweet roots”.
• Therapeutic use of licoricedates back to the Roman Empire. The Greek physician Hippocrates (460 BC) and botanist Theophratus (371 BC) extolled its uses.
• Licorice root contains a variety of compounds, including triterpenoids, polyphenols, and polysaccharides (starches, mannose, and sucrose). Polyphenols include certain phenolic acids, such as liquiritin, flavones and flavans; chalcones; and isoflavonoids, such as glabridin.
• The activity of licorice on the immune system has been described as "nonspecific" by most investigators. This means licorice stimulates, activates or promotes an immune response in multiple ways.
S. Rao Chavali et al report on Glycyrrhizic acid that the experimental data suggest that different components in the Quillaja saponin preparations may have selective effects on various subtypes of cell populations. Glycyrrhizic acid has the most profound immunomodulatory activity in vitro. Activation, proliferation and differentiation steps of lymphocytes appear to be mediated by secretion of soluble factors.

Minerals

The present invention combines above mentioned rasayana herbal medicines preferably around 5 – 10 rasayana herbs in the form of powders / Ghan with 1 - 4 minerals preferably in the form of bhasma or in colloidal or in nano particulate form in such a way that they can be combined in two or more preparations and administered together, simultaneously or sequentially to impart / boost immunity to humans and mammals thus building ability to identify and resist large numbers of infectious and potentially harmful microorganisms, enabling the body to prevent or resist diseases and inhibit organ and tissue damage. The preparations so combined and administered can help patients.

10. Calcium
A study conducted by Razzell et al (2013) in the University of Bristol on cellular processes underlying the body's response to healing has shown for the first time how a flash of calcium is the very first step in repairing damaged tissue. The findings could lead to new therapies that speed up the healing process following injury or surgery.

11.Calcined Zinc –
Due to antioxidant effects of Zinc it protects against ROS and RNS. Zinc helps modulate cytokine release and induces proliferation of T cells and helps to maintain skin and mucosal membrane integrity.
Silvia Maggini et al referring previously conducted research report that Zinc has a central role in cellular growth and differentiation of immune cells. It is essential for intracellular binding of tyrosine kinase to T cell receptors, required for T lymphocyte development and activation. Zinc supports Th1 response.
Melinda A. Beck reports that Zn is perhaps one of the most studied trace elements with respect to its effect on the host immune system and that Zinc deficiency has been noted to result in increased susceptibility to infectious disease. Suboptimal Zinc status has also been associated with decreased T-cell function and antibody responses. If the Zinc deficiency is corrected, immune status is restored . Zinc is an essential cofactor for thymulin, a peptide hormone that plays a key role in T-cell maturation.
Additionally, minerals Silver and / or gold can be added in bhasma or in colloidal or in nano particulate form.

Process of Preparation
1. Rasayana Herbs
Herbal medicines are preferably provided in ghana form. The process of preparing Ghan form can be from water / aqueous extract or from super critical extraction process. Any other extraction process used for preparing ghan can also be adopted. The processes are provided under Figures 1 - 3.
The administration is by oral route and accordingly oral solid and liquid compositions are designed.

2. Bhasma preparation
Minerals calcium and zinc can be converted into Bhasma form by usual reported processes. Alternatively, shankh bhasm and Jasad bhasm can be incorporated. Preferably, the particle size of calcium and zinc in bhasma are such that 90 % particles are below 50 µM, more preferably below 15 µM and most preferably below 1 µM. Alternatively, colloidal or nano particulate forms are used. The particle size of mineral in these forms is found to be crucial.
Zinc content can vary in bhasma form. In an embodiment, zinc content is around 70 %. Calcium content in an embodiment of bhasma is around 40 %.

3. Preparation of compositions:
Various compositions or formulations combining 3 – 10 rasayana herbal medicines and from 1 – 4 minerals are prepared in different dosage forms such as tablet, capsule, caplets, coated tablets, multi-layered tablets, minitablets, pill, powder, granules, seeds, pellet, extrudates, spheronized material, compacts, particulate preparation, dispersion, dispersible tablet, fast disintegrating tablet, lozenge, sublingual or buccal tablets, powder mixture, syrup, suspension, powder for oral solution, powder for oral suspension injectables, liquid, solution, subcutaneous, intravenous, intramuscular composition.

The present invention aims at providing various compositions containing herbal medicines and minerals.

The ghan of different rasayana herbs are mixed and blended further with one or more mineral in form of bhasma (or alternative forms). Suitable pharmaceutically acceptable excipients / adjuvants are added and blended further. Excipients include one or more of diluents, binders, glidants, lubricants and disintegrants. Generally excipients occupy from 2 – 40 % preferably from 5 – 30 % of the final compositions.
One of the preferred solid dosage form is a capsule dosage form. It can contain a mixture of herbal powders and minerals in the form of bhasma or in colloidal or in nano particulate form and necessary adjuvants.
The other dosage form is a tablet containing a mixture of herbal powders and minerals in the form of bhasma or in colloidal or in nano particulate form and necessary adjuvants.
Yet one more dosage form is a taste masked syrup or suspension containing dissolved / partially dissolved or finely suspended mixture of herbal powders and minerals in the form of bhasma or in colloidal or in nano particulate form and necessary adjuvants.

Analysis of herbomineral compositions
The rasayana herbs / herbomineral compositions will be subjected to following analytical processes to estimate active contents or marker compounds from rasayana herbs / compositions.
Table 2: Parameter to be analyzed.

Product Standardization parameter
1 Ginger Gingerols Content by HPLC
2 Vavding Embelin by HPLC
3 Licorice Glycyrrhizin content by Gravimertric
4 Haritaki Chebulinic acid, Chebuloside II by HPLC
5 Guduchi Cordiofolioside A by HPLC
6 Shatavari Shatawarin IV Content by HPLC
7 Amla Gallic and Ellagic Acid By HPLC
8 Ashwagandha Withanolides by Gravimetric
9 Long pepper Piperine Content by HPLC

Clinical trials for Covid-19 patients
COVID-19 (previously termed as 2019-nCoV), a novel coronavirus disease with high mortality, emerges as a pandemic disease . In December 2019, a series of acute atypical respiratory disease occurred in Wuhan, China. This rapidly spread from Wuhan to other areas. It was soon discovered that a novel coronavirus was responsible. The novel coronavirus was named as the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, 2019-nCoV) due to its high homology (~80%) to SARS-CoV, which caused acute respiratory distress syndrome (ARDS) and high mortality during 2002–2003.
The disease caused by this virus was called Coronavirus disease 19 (COVID-19) and a pandemic was declared by the World Health Organization (WHO). COVID-19 has been impacting a large number of people worldwide, being reported in more than 200 countries and territories.
SARS-CoV-2 virus primarily affects the respiratory system, although other organ systems are also involved. Lower respiratory tract infection related symptoms including fever, dry cough and dyspnea were reported in the initial case series from Wuhan, China. In addition, headache, dizziness, generalized weakness, vomiting and diarrhoea were observed. It is now widely recognized that respiratory symptoms of COVID-19 are extremely heterogeneous, ranging from minimal symptoms to significant hypoxia with Acute Respiratory Distress Syndrome (ARDS). In the report from Wuhan mentioned above, the time between the onset of symptoms and the development of ARDS was as short as 9?days, suggesting that the respiratory symptoms could progress rapidly. This disease could be also fatal. A growing number of patients with severe diseases have continued to succumb worldwide. Epidemiological studies have shown that mortalities are higher in elder population and the incidence is much lower in children.
Current medical management is largely supportive with no targeted therapy available. More treatment modalities are being tested in clinical trials in many countries. A large number of countries have implemented physical distancing and lockdown to mitigate further spread of the virus.
Due to sudden advent and rapid spread of infection, no antiviral drug has been found to be effective since its onset.
Clinical trials are conducted at Quest Clinical Services, a Pune based CRO.
The present inventors have invented various formulation which are effective in treating Covid-19. These formulations are reported in our previously filed provisional patent application numbered 202021031642 dated 23-07-2020.
The investigational product (IP) is in the form of a capsule hereinafter Capsule IP or Cap IP.
To check the efficacy and safety of this combination on the Covid positive patients, the inventors have done a clinical trial as an add-on therapy for the Covid positive patients keeping in mind that:
1. While there is no medicine for COVID-19 as of now, enhancing body’s defence system (immunity) should play an important role in maintaining optimum health and the IP (study drug: IP) might possibly play an important role in enhancing immunity.
2. Role of study drug / combination of rasayana herbal medicines or herbal medicines as an antiviral is also tested.
3. Role of study drug / combination of rasayana herbal medicines or combination of herbal medicines in relieving symptoms and in the hospital.

Details of the clinical trial in Covid-19 positive patients
1. Study Title: A double blind randomized controlled trial is planned to assess safety and efficacy of Cap IP in COVID-19 positive patients with mild to moderate severity for early recovery, in restoring respiratory health and in improvement in innate immunity.
2. CTRI Number: CTRI/2020/07/026570
3. Site: Yashwantrao Chavan Memorial Hospital, Pimpri, Pune, India
4. Date of EC approval: 12th June 2020
5. Date of First Enrollment: 11th August 2020
6. Date of Last Enrollment: 3rd September 2020
7. Enrollment Duration: 23 days (18 working days)
8. Total no. of enrollments: 72
Figure 4 provides number of patients screened, enrolled and given drug i.e. Cap IP or placebo in the present study.
Figure 5 provides number of patients enrolled for the study over time and gender distribution of patients enrolled.
Average age of enrolled patients = 46 years.

9. Number of Consent Withdrawals / Dropouts: 6
10. SAE reported: One
Efficacy of the study drug was measured on three major parameters:
1. Clinical improvement
2. Viral load decline
3. Immunological improvement
Following were objectives of the trial:
• To assess efficacy of Cap. IP in boosting innate immunity of patients with COVID-19 infection
• To assess efficacy of Cap. IP in restoring respiratory health
• To see efficacy of Cap. IP in early recovery of patients and decline in Viral Load
• To assess safety of Cap. IP.
Study Design
• This was a double blind randomized study.
• A total of 72 COVID-19 patients were enrolled in the study.
• Those fulfilling eligibility criteria and consenting in writing were enrolled in the study.
• All the patients enrolled in the study got the Investigational Product (Cap. IP), or Placebo two times daily.
• Patients underwent laboratory investigations upon enrolment and on last visit. RTqPCR for COVID was done on day 4 (+ 1 day).
• Patients and doctors treating were not knowing what the participating subject had received. Subjects were given coded bottles, containing dose for 30 days.
• No medicine which his/her physician was giving was stopped. No modifications was suggested in their diet and daily routine physical activities.
• Thus, patients enrolled in the study got the IP / Placebo in addition to the standard of care which they were already receiving at the time of enrolment.

Study period for enrolled patients: 30 days:
• Enrolment (day 0): After Informed Consent Process, Demographics and other information for CRF was recorded. Investigations: COVID 19 RTqPCR, Lymphocyte subset analysis (TH1, TH2, Th17, IL6, NK Cells and CD markers); Immunoglobulin IGG (Serum); Immunoglobulin IGM (Serum); Liver Function Test and Kidney Function Test was done. Cap. bottle was given to the patients with the dose lasting for 30 days.
• Follow-up 1: Day (4±1): COVID-19 RT PCR (quantitative) test was conducted for all subjects on Day (4±1 day). CRF was filled for knowing the overall status like any hospital stay or isolation etc.
• Follow up 2 (15th day+ 2 days): This follow up was either clinical or telephonic. CRF was filled for knowing the overall status.
• Follow up 3 (30th day+ 2 days): Patients were followed up at day 30. This visit was in a nature of telephonic follow up. Patient did not visit site but blood investigations were carried out by home visit. Investigations were done outside the site, considering that the site is COVID specific hospital. The investigations were; Lymphocyte subset analysis (TH1, TH2, Th17, IL6, NK Cells and CD markers); Immunoglobulin IGG (Serum); Immunoglobulin IGM (Serum); Liver Function Test and Kidney Function Test was also done. All the unused IP will be collected.
• Sponsor provided 10 codes (5 from each, IP and placebo, not mentioning which is placebo and IP). Sr. Ferritin, CRP and D- Dimer was done for same patients on day 0 and day 4.

Figure 6 provides a flow chart indicating steps from screening, enrollment and first, second and third follow up after administration of Cap IP or placebo.
Methods:
Patients attending COVID set-ups (YCM Hospital, Pimpri, Pune) of the investigators were screened for their participation in the study. Patients were COVID-19 positive, diagnosed recently. No discretion for enrolment was made based on gender, color, religion and cast. Information about the study was shared with the patients and their eligibility was checked during screening. Those fulfilling this requirement and who were willing to participate were consented. All questions were answered to the patients’ satisfaction. Patient were considered as enrolled only after consenting procedure was successfully done.
Patients were counselled about administration of Cap IP (dose, timing, method etc.).
Follow-Up:
After enrolment (Day 0), there was one clinical and the next two: either telephonic or clinical follow-ups: first follow-up visit at day 4 (+1 day), second follow-up visit at day 15 (+2 days) and third follow-up visit at day 30 (+2 days).
Figure 6 provides a flow chart indicating steps from screening, enrollment and first, second and third follow up.
Patients could call the study coordinator anytime for the queries if any.

Study Design:
This was a double blind, prospective study to determine safety and efficacy of Cap. IP in COVID-19 positive patients.
After enrolment, they got the IP / Placebo in addition to their regular treatment they might be receiving for their condition.
A total of 72 patients were enrolled in the study in two groups.
The reason behind this sample size is that, one can apply paired t test with adequate power for an exploratory study.

Table 3: Sample size: Cross- sectional, Cohort and Randomized Clinical Trials

Two-sided significance level (1 alpha) 95
Power (1-beta. % chance of detecting) 80
Ratio of sample size, Unexposed/ exposed 1
% of Unexposed with outcome 5
% of Exposed with outcome 35
Odds ratio 10
Risk/ Prevalence ratio 7
Risk/ Prevalence 30

Kelsey Fleiss
Sample Size Exposed 28 27
Sample Size Non exposed 28 27
Total Sample Size 56 54

References:
Kelsey et al, Methods in Observational Epidemiology, 2nd edition, table 12-15, Fleiss, Statistical Methods For Rates and Proportions, formulas 3.18 and 3.19.

Results are rounded up to the nearest integer
Results from Open Epi, Version 3, open source calculator –SSC cohort.
Considering dropouts, 30 in each arm were considered earlier.
Study Population:
COVID-19 positive subjects.
Subject Eligibility:
(a) Inclusion Criteria
1. Age > 18 years
2. All sexes
3. Case definitions for inclusion in the study will include mild to moderately severe cases as defined in the Guidance on appropriate treatment of suspect/confirmed case of COVI19 issued by Ministry of Health and Family Welfare, Govt of India on 07 Apr 2020.
Mild: Cases presenting with fever and/or upper respiratory tract illness (Influenza like Illness, ILI); Moderate: Pneumonia with no signs of severe disease (Respiratory Rate 15 to 30/minute, SpO2 90%-94%). OR
4. Laboratory confirmed SARS CoV-2 infection within last 10 days or SARS CoV-2 test result pending with a high clinical suspicion as defined by: Cough of more than 10 days duration OR
5. Bilateral pulmonary infiltrates on chest X Ray / CT scan or new hypoxaemia defined as SpO2 <94% on room air, where no alternative explanation for respiratory symptoms can be given.
(b) Exclusion Criteria
1. Pregnant or lactating women
2. Symptoms of acute respiratory tract infection for more than seven days
3. More than 48 hours have elapsed between meeting inclusion criteria and enrolment
4. Subject is also a participant of any other clinical trial
5. Serious / long-standing co-morbid conditions

Table 4: Study Assessment Table
Visit? Screening Visit
Enrolment 1st Follow up
2nd Follow up
3rd Follow up

Study Procedure (Day -2 to Day 0) Day 0 Day 4th (+1) Day 15th (+2) Day 30th (+2)
ICF X X
I/E Criteria X X
Demographics X
Vitals and Physical Examination X X X X
Medical History X
Questionnaire X X X X
Investigations X*# X# X*
Bottle Dispensing X X
IP / Placebo Accountability X X X
AE/SAE X X X X X

Investigations –
*TH1, TH2, Th17, IL6, NK Cells and CD markers; Immunoglobulin IGG (Serum); Immunoglobulin IGM (Serum); Liver Function Test and Kidney Function Test
Around 2 ml of blood will be stored for additional biomarker in Immunology and Virology.
# Nasopharyngeal Swab for Quantitative RTPCR
A Small group: Sr. Ferritin, CRP and D-Dimer will be done on day 0 and 4.
Results and Interpretation:
1. Viral Load:
In prior analyses of the SARS-CoV-1 outbreak, viral load within the nasopharynx was associated with worsened disease severity and increased mortality23-24. However, it is also clear that there are significant differences between SARS-CoV-1 and 2. There was a statistically significant survival probability between those with a high (defined as greater than 5.557 log10/ml viral load) and those with lower viral load (<5.19 log10/ml) 25. Detectable plasma viremia using a qualitative viral detection assay may correlate with disease severity26.
Studies to date have been hampered by the lack of viral load quantification. In present study quantitative viral loads were performed on Day 1 and Day 4+
Oro and nasopharyngeal swab of 66 patients was collected for quantitative RT-PCR on day 4. The test provided results not of a ‘surrogate marker’- Ct value, but a concrete one: Viral Load (copies / mL).
Brief methodology for quantitative estimation of SARS-CoV-2 viral load in respiratory samples:
Sample collection and nucleic acid extraction:
Clinical samples for SARS-CoV-2 testing consisted of nasopharyngeal and oropharyngeal swabs collected from each patient and transported in Viral Transport Medium (VTM). After receiving the samples in the laboratory, the tubes were vortexed and centrifuged at 2500 rpm for two minutes. Two hundred microlitres of the sample was used as input to Thermo Fisher Scientifics’ KingFisherTM Flex Purification System. Briefly, samples were lysed and the RNA was eluted in a 50uL elution buffer by a magnetic bead-based elution method using the MagMAXTM Viral/Pathogen II (MVP II) Nucleic Acid Isolation Kit.

Quantitative estimation of viral load using qRT-PCR:
Three viral loci RdRP, N gene and E gene and a human gene as internal control (RNaseP) were detected using GenePath Diagnostics’ indigenously developed qRTPCR assay, CoViDx One v2.1.1 keeping appropriate controls and standards. Ten microlitres of extracted nucleic acid was used as an input for the qRT-PCR. Quantitative estimation for each genomic target was performed using standards, for which the number of genome copies were already known. The threshold cycle (Ct value) for each genomic target was extrapolated to the viral load of standards to estimate the number of copies/uL which were then converted to copies/mL for each gene for each sample. Average number of copies/ ml was calculated for each sample that accounts for the approximate viral load in the sample. Ct values for control human gene (RNaseP) ensured the adequacy of sample and served as control for nucleic acid extraction and nucleic acid amplification.
Following is the statistics:

Table 5: For Drug Arm:
Descriptive
Statistic Std. Error
Viral Load Before Mean 117593314.663132 56521251.1890938
95% Confidence Interval for Mean Lower Bound 2161520.163975 -
Upper Bound 233025109.162290 -
5% Trimmed Mean 63883032.025344 -
Median 662081 -
Variance 99034206915399600.000 -
Std. Deviation 314697008.1132000 -
Minimum 297.0000 -
Maximum 1.2365E+09 -
Range 1236492393.0000 -
Interquartile Range 19428808.0000 -
Skewness 2.980 .421
Kurtosis 8.130 .821
Viral Load After Mean 2955210.522266 1323966.9597615
95% Confidence Interval for Mean Lower Bound 251309.267292 -
Upper Bound 5659111.777239 -
5% Trimmed Mean 1693734.539180 -
Median 48963 -
Variance 54339543826743.300 -
Std. Deviation 7371536.0561245 -
Minimum 0.0000 -
Maximum 3.5297E+07 -
Range 35296626.3971 -
Interquartile Range 1239483.0000 -
Skewness 3.378 .421
Kurtosis 12.589 .821

N Mean Rank Sum of Ranks
Viral Load After - Viral Load Before Negative Ranks 23a 17.52 403.00
Positive Ranks 8b 11.63 93.00
Ties 0c
Total 31
a. Viral Load After < Viral Load Before
b. Viral Load After > Viral Load Before
c. Viral Load After = Viral Load Before

Test Statistics
Viral Load After - Viral Load Before
Z -3.037b
Asymp. Sig. (2-tailed) .002
a. Wilcoxon Signed Ranks Test
b. Based on positive ranks.

P value is .002 (highly significant) for Drug arm
Table 6: For Placebo Arm:
Placebo
Statistic Std. Error
Viral Load Before Mean 12409445.67 5525858.632
95% Confidence Interval for Mean Lower Bound 882706.55
Upper Bound 23936184.79
5% Trimmed Mean 9026436.48
Median 385670.00
Variance 641237386013617.000
Std. Deviation 25322665.460
Minimum 735
Maximum 85986651
Range 85985916
Interquartile Range 13121040
Skewness 2.376 .501
Kurtosis 4.875 .972
Viral Load After Mean 69693979.492821 47868373.8000342
95% Confidence Interval for Mean Lower Bound -30157698.534105
Upper Bound 169545657.519747
5% Trimmed Mean 33896420.012919
Median 66845.000000
Variance 48119005415455800.000
Std. Deviation 219360446.3330980
Minimum 365.0000
Maximum 7.8974E+08
Range 789739089.0000
Interquartile Range 824146.5000
Skewness 3.020 .501
Kurtosis 7.980 .972

N Mean Rank Sum of Ranks
Viral Load After - Viral Load Before Negative Ranks 14a 11.57 162.00
Positive Ranks 7b 9.86 69.00
Ties 0c
Total 21
a. Viral Load After < Viral Load Before
b. Viral Load After > Viral Load Before
c. Viral Load After = Viral Load Before

Placebo
Viral Load After - Viral Load Before
Z -1.616b
Asymp. Sig. (2-tailed) .106
a. Wilcoxon Signed Ranks Test
b. Based on positive ranks.

P Value is not significant for placebo arm.
Standard of care was not stopped / interfered in either of the arms. Hence the additional benefit the drug arm got, we can certainly contribute to the study drug.
2. Clinical Assessment:
Patients enrolled were of mild and moderate category. On the clinical front, although it’s a qualitative parameter, visible difference was noticed in favour of the drug arm on day 4 itself.
Numerical Rating Scale was used for overall disease severity. 0 was considered for normal, 1 to 3 for mild, 4 to 6 for moderate and 7 to 10 for severe severity of the disease. Patients were asked to rate the severity. This was to quantify the qualitative clinical presentation of COVID 19. Majority of the patients scored below 7 at baseline.
Table 7: Numerical rating scale
Table 7A: Whole data
Whole data
Mean N Std. Deviation Std. Error Mean
Pair 1 Before 4.32 63 1.075 .135
After 2.49 63 1.243 .157

Table 7B: Drug
Drug
Mean N Std. Deviation Std. Error Mean
Pair 1 Before 4.42 31 1.057 .190
After 1.81 31 1.078 .194

Table 7C: Placebo
Mean N Std. Deviation Std. Error Mean
Pair 1 Before 4.22 32 1.099 .194
After 3.16 32 1.019 .180

P value is statistically significant for both the groups, however reduction in the mean for drug group was very prominent compared to the placebo group.
Table 8: Numerical rating scale
Numerical Rating Scale Before After Difference
Drug 4.42 1.81 2.61
Placebo 4.22 3.16 1.06

The decline in % in the drug arm was 59% whereas it was 25% for placebo arm. This points out that the decline in the subjective feeling of illness and symptoms was prominent in four days compared to the same in patients receiving placebo.
Blood Biomarkers:
For majority of the markers (i.e. except the inflammatory and acuity representing markers viz. CRP, D-Dimer, Sr. Ferritin, all other markers were done on day 0 (enrolment day) and on day 30.
Almost all the patients were discharged before day 30, and patients were reluctant to come to Covid center again at day 30.
Study team faced difficulties, and only around 50% of patients were ready for blood withdrawal at day 30. The reasons for difficulties were fear of hospitalization, reinfection, family pressures, feeling better so not willing for blood donation. Due to less number of patients, it was not possible to apply a statistical test with good confidence. Hence below mentioned are the averages (means) of the collected data at day 0 and day 30 (for immunology markers). Absolute difference certainly provide good clue in favour of drug and hints toward a bigger study involving more subjects.
1. Th1, Th2 and Th17
The Th1 pathway is often portrayed as being the more aggressive of the two and release various proinflammatory cytokines like IL1, IL2, IL12, TNF-a, IFN-?, etc. while the Th2 pathway is seen to release various anti-inflammatory cytokines like IL4, IL5, IL6, IL10, etc. which are also involved in several allergen-specific and related IgE-based events like development of airway eosinophilia, the induction of airway smooth muscle contraction, mucus production, etc. and also predisposing to several systemic autoimmune diseases.
Most recent data demonstrated that COVID-19 might affect lymphocytes, especially T lymphocytes. The absolute number of T lymphocytes, CD4+ T cells, and CD8+ T cells decreased in nearly all the patients and was markedly lower in severe cases .
Adaptive host immunity against viral pathogens is key to eliminate the viral particles from the body, and it is the main path of defence, recovery, and prevention through a successful vaccination program. Until then the initial antiviral immunity mostly involves innate immunity and activation of T-helper cells or the Th1 response. One of the main immunological features of the SARS-CoV-2 infection is the presence of lymphopenia and exuberant release of acute phase reactants such as C-reactive protein and the cytokine interleukin-6 (IL-6). Elevated levels of IL-17 are the primary stimulus that shifts an immune response from a Th2 to a Th1 response pattern. Increases in Th1/Th2 ratio lead to the release of cytokines IL-1ß, IL-2, IL-12, TNFa, and interferon gamma (IFN?).(2) Excessive release of inflammatory cytokines, such as IL-1ß, TNFa, IL-6, and IL-8, while necessary to eliminate the viral pathogen, can create a clinical condition known as cytokine storm.
Further downstream activation of neutrophils also occurs during this cytokine storm and leads to the release of neutrophil extracellular traps (NETs).
Drug Arm:
Mean Absolute Th1 Count was 267 at day 0 which increased by 188 and became 455 on Day 30.

Placebo Arm:
Mean Absolute Th1 Count was 261 at day 0 which increased by 96 and became 357 on Day 30.
The increase in Th1, as can be noticed was almost 100% more in Drug arm than that in the Placebo arm.
Drug Arm:
Mean Absolute Th2 Count was 247 at day 0 which increased by 347 and became 594 on Day 30.
Placebo Arm:
Mean Absolute Th2 Count was 181 at day 0 which increased by 287 and became 468 on Day 30.
Drug Arm:
Mean Absolute Th17 Count was 0.2 at day 0 which increased by 0.6 and
became 0.8 on Day 30.
Placebo Arm:
Mean Absolute Th17 Count was 0.06 at day 0 which increased by 0.31 and became 0.37 on Day 30.

Table 9: Comparison of Th1, Th2 and Th17

Table 9A: Comparison of Th1
Th1 Before After Difference
Drug 267 455 188
Placebo 261 357 96

Table 9B: Comparison of Th2

Th2 Before After Difference
Drug 247 594 347
Placebo 181 468 282

Table 9C: Comparison of Th17

Th17 Before After Difference
Drug 0.2 0.8 0.6
Placebo 0.06 0.37 0.31

2. Total Leucocyte Count (TLC):
Higher the leucocyte count higher is the chances of severity of COVID. Zhao K et. al. Reports that patients with leucocytosis were found to be more severely ill as compared to others and would likely be admitted in ICU and need mechanical ventilation.
Also confirmed COVID-19 patients with leucocytosis exhibited significantly raised levels of procalcitonin, CRP and IL-6 in the serum when compared with the patients with non-increased leukocyte count. The patients with increased leukocyte count were significantly older, and were more likely to have fever and underlying chronic diseases, thereby suggesting that the older patients with COVID-19 who had underlying chronic disorders are more likely to develop leukocytosis.
The confirmed COVID-19 patients with increased leukocyte count receiving antibiotics still had a considerable number of critical illness, with a high admission to an ICU and a high mortality rate

Table 10: Total Leucocyte Count

TLC Before After Difference
Drug 7240 7607 367
Placebo 6257 7200 943

Drug Arm:
Mean Total Leucocyte Count was 7240 at day 0 which increased by 367 and became 7607 on Day 30.
Placebo Arm:
Mean Total Leucocyte Count was 6257 at day 0 which increased by 943 and became 7200 on Day 30.
The placebo group had twice the increase in leucocytes as compared to the trial group.

3. Absolute B Cells:
Humoral immunity depends on the ability of B cells to successfully transform into plasmocytes and produce antibodies. The production of antibodies to COVID-19 is largely dependent upon the response by Th cells, which typically occurs one week after COVID-19 symptoms begin.
The COVID-19 antibody response has been identified to begin between 4 and 8 days after symptoms begin; however, the neutralizing activities of these antibodies do not begin until at least 2 weeks after symptom onset.
Although the long-term stability of these neutralizing antibodies has not yet been confirmed, they have the potential, with the assistance of memory B cells, to prevent against reinfection if a patient encounters SARS-CoV-2 again in the future. Several monoclonal antibodies have been developed from the memory B cells of former COVID-19 patients as potential therapies.
Although the neutralization capabilities of certain antibodies are believed to be effective against SARS-CoV-2 and thereby have protective effects, B cells can also produce non-neutralizing antibodies.
Table 11: Absolute B cells
Absolute B Cells Before After Difference
Drug 256 437 181
Placebo 193 318 125

Drug Arm:
Mean Absolute B cell Count was 256 at day 0 which increased by 181 and became 437 on Day 30.
Placebo Arm:
Mean Absolute B cell Count was 193 at day 0 which increased by 125 and became 318 on Day 30.

4. Absolute T Cells:
Once an individual becomes infected with SARS-CoV-2, the virus will remain in its incubation period for between 4 and 5 days before the patient begins to develop symptoms. Approximately one week after COVID-19 symptoms begin, both B and T cell responses can be detected in the blood.
Early studies conducted on some of the first COVID-19 patients found that mononuclear cells, which most likely included monocytes and T cells, accumulated within the lungs, whereas low levels of hyperactive T cells were identified in the peripheral blood. The presence of T cells in such low levels within the blood suggests that rather than remain within the bloodstream, T cells travel from the blood into the infected organs to mitigate the immune response.
More specifically, this response involves the there is a massive T cell response to the acute infection, in which CD4+ and CD8+ T cells are predominantly involved. While this early T cell response appears to be protective, the ability of this specific immune response to prevent infection in humans has not been fully evaluated.
Table 12: Absolute T cells
Absolute T Cells Before After Difference
Drug 837 1721 884
Placebo 711 1513 802

Drug Arm:
Mean Absolute T cell Count was 837 at day 0 which increased by 884 and became 1721 on Day 30.
Placebo Arm:
Mean Absolute T cell Count was 711 at day 0 which increased by 802 and became 1513 on Day 30.

5. Absolute CD3+CD4+T helper Cells:
Human peripheral CD4+ T cells can be characterized as naive (CCR7+CD45RA+), central memory (CCR7+CD45RA–) and effector-memory (CCR7–CD45RA–) cells that respond differently during antigen re-exposure.
Patients recovered from SARS showed persistent memory CD4+ T cells that could be potentiated by spike protein.
Mouse experiments also demonstrated boosting memory CD4+ T cells can protect mice from SARS and MERS infection. Thus, better understandings of these memory CD4+ T cells in COVID-19 patients could help us develop long-term host protection to this disease.
CD4+ T follicular helper (Tfh) cells are critical for high-affinity antibody response and successful vaccination during infection.
Both cytotoxic and suppressor T cells (CD3+CD8+) in patients with COVID-19 were below normal levels in severe cases and negatively correlated with the levels of TNF-a, IL-6, and IL-10.
Decreased number of T cells observed in COVID-19 patients is probably due to high serum concentration of TNF-a, IL-6, and IL-10 as well as negative regulating impact on T cell survival or proliferation; therefore, promoting a decrease in T cells by these cytokines in individuals with severe disease is probable
Table 13: Absolute CD3+CD4+T helper Cells
Absolute CD3+CD4+T helper Cells Before After Difference
Drug 515 1056 541
Placebo 400 835 435

Drug Arm:
Mean Absolute CD3+CD4+T helper Cells Count was 515 at day 0 which increased by 541 and became 1056 on Day 30.
Placebo Arm:
Mean Absolute CD3+CD4+T helper Cells Count was 400 at day 0 which increased by 435 and became 835 on Day 30.

6. Absolute CD3+CD8:
CD8+ T lymphocytes mediate adaptive cytotoxic T cell responses. Cytotoxic cells can contribute to virus control by eliminating infected cells. CD3+, CD4+ and CD8 + T lymphocyte subsets were decreased in COVID-19-infected patients

Table 14: Absolute CD3+CD8:

Absolute CD3+CD8 Before After Difference
Drug 300 607 307
Placebo 271 571 300

Drug Arm:
Mean Absolute CD3+CD8 Cells Count was 300 at day 0 which increased by 307 and became 607 on Day 30.
Placebo Arm:
Mean Absolute CD3+CD8 Cells Count was 271 at day 0 which increased by 300 and became 571 on Day 30.

7. Absolute T regulatory:
T regs ensure that inflammation is kept under control when there is an immune response. The current evidence suggests that the level of peripheral Tregs is prominently reduced in severely ill COVID-19 patients compared with mild patients. Though the reasons for reduced frequency of Tregs in peripheral blood are not completely understood, one of the possibilities is that Tregs might have migrated to the lungs to prevent tissue damage. Tregs and their functions are compromised in severe COVID-19 patients, engendering unrestrained immune cell activation resulting in damaged lungs in severe COVID-19 patients

Table 15: Absolute T regulatory

Absolute T regulatory Before After Difference
Drug 6.4 4.8 -1.6
Placebo 5.9 3.6 -2.3

Drug Arm:
Mean Absolute T regulatory Cells Count was 6.4 at day 0 which reduced by 1.6 and became 4.8 on Day 30.
Placebo Arm:
Mean Absolute T regulatory Cells Count was 5.9 at day 0 which reduced by 2.3 and became 3.6 on Day 30.
There is less reduction of T regs in the drug group as compared to the placebo group suggesting a good inflammatory control in the subjects of the drug group.

8. Absolute NK Cells:
NK cells generally tends to be lower in severe cases.
Preliminary studies in COVID-19 patients with severe disease suggests a reduction in NK cell number and function, resulting in decreased clearance of infected and activated cells, and unchecked elevation of tissue-damaging inflammation markers.
SARS-CoV-2 infection skews the immune response towards an overwhelmingly inflammatory phenotype.
Restoration of NK cell effector functions has the potential to correct the delicate immune balance required to effectively overcome SARS-CoV-2 infection.

Table 16: Absolute NK Cells

Before After Difference
Drug 9.9 28.2 18.3
Placebo 10.4 17.5 7.1

Drug Arm:
Mean Absolute NK Cells Count was 9.9 at day 0 which increased by 18.3 and became 28.2 on Day 30.
Placebo Arm:
Mean Absolute NK Cells Count was 10.4 at day 0 which increased by 7.1 and became 17.5 on Day 30.
There is an increase in NK cell count in the drug group as compared to the placebo group

9. Absolute Neutrophil Count:
Increased neutrophil counts were reported in peripheral blood of severe and non-surviving COVID-19 patients. Neutrophilia predicts poor outcomes in patients with COVID-19, and research also indicated the neutrophil-to-lymphocyte ratio (NLR) is an independent risk factor for severe disease. Excessive neutrophils lead to tissue injury by oxidative burst, phagocytosis, and the formation of neutrophil extracellular traps (NETs). Neutrophilia and excessive NETs may contribute to cytokine release and respiratory failure
Table 17: Absolute Neutrophil Count

Absolute Neutrophil Count Before After Difference
Drug 4615 4046 -569
Placebo 4457 3986 -471

Drug Arm:
Mean Absolute Neutrophil Count was 4615 at day 0 which reduced by 569 and became 4046 on Day 30.
Placebo Arm:
Mean Absolute Neutrophil Count was 4457 at day 0 which reduced by 471 and became 3986 on Day 30.

10. Absolute Lymphocyte Count:
Lymphocytopenia in patients admitted to the Intensive Care Unit (ICU) is a marker of disease severity. Patients with lymphocytopenia were more likely to develop an acute kidney injury (AKI). It is a good prognostic factor in determining the clinical course and disease severity of a patient admitted to the hospital for Covid-19
Table 18: Absolute Lymphocyte Count
Absolute Lymphocyte Count Before After Difference
Drug 1654 2846 1192
Placebo 1250 2343 1093

Drug Arm:
Mean Absolute Lymphocyte Count was 4615 at day 0 which reduced by 569 and became 4046 on Day 30.
Placebo Arm:
Mean Absolute Lymphocyte Count was 4457 at day 0 which reduced by 471 and became 3986 on Day 30.

11. IgG and IgM:
IgM can be detected as early as 3 days after infection and provides the first line of humoural immunity defence, after which high affinity IgG responses are initiated and play a key role in long term immune memory. Therefore, the detectable levels of IgM and IgG antibodies could provide information regarding serological convention over the disease course, as the detection of IgM antibody indicates a recent exposure to SARS-CoV-2 and the detection of IgG antibody in the absence of detectable IgM antibody indicates prior virus exposure.
The increased IgM level in the deceased case group might be related to the higher disease severity in these patients and indicate a poor prognosis. Alternately, cytokine storm, severe immune dysfunction and other co-mobidities might be the important risk factors in these cases.
It is widely accepted that the IgM antibody response provides early-stage defence during viral infections prior to the development of the class-switched, high-affinity IgG response for long-term immunity and immunological memory.
Figure 7 provides antibody concentrations of IgG and IgM in serum RU/ml over period after onset of symptoms.

Table 19: Typical phenomenon of IgG and IgM in COVID’19 patients.

Table 19A: IgG
IgG Before After Difference
Drug 1344 1335 -9
Placebo 962 1132 170

Drug Arm:
Mean IgG was 1344 at day 0 which reduced by 9 and became 1335 on Day 30.
Placebo Arm:
Mean IgG was 962 at day 0 which increased by 170 and became 1132 on Day 30.
Table 19B: IgM
IgM Before After Difference
Drug 110 93.3 -7
Placebo 47 113 66

Drug Arm:
Mean IgM was 110 at day 0 which reduced by 7 and became 93.3 on Day 30.
Placebo Arm:
Mean IgM was 47 at day 0 which increased by 66 and became 113 on Day 30.

12. COVID Antibodies:
Table 20: COVID Antibodies:

COVID Antibodies
Drug 5.8
Placebo 4.95

Drug Arm:
Mean COVID Antibodies detected on Day 30 was 5.8
Placebo Arm:
Mean COVID Antibodies detected on Day 30 was 4.95
There was almost 20% more antibodies in drug arm compared to placebo.

Inflammatory and Other Markers:
1. C-Reactive Protein (CRP):
CRP levels are correlated with the level of inflammation, and its concentration level is not affected by factors such as age, sex, and physical condition. CRP levels can be used for early diagnosis of pneumonia , and patients with severe pneumonia had high CRP levels. It is an important index for the diagnosis and assessment of severe pulmonary infectious diseases. The study showed that CRP levels and the diameter of the largest lung lesion increased as the disease progressed. CRP levels were positively correlated with lung lesion and disease severity. This suggests that in the early stage of COVID-19, CRP levels could reflect lung lesions and disease severity .
A significant association was observed between CRP concentrations and the aggravation of non-severe patients with COVID-19, and the authors proposed CRP as a suitable marker for anticipating the aggravation probability of non-severe COVID-19 patients, with an optimal threshold value of 26.9?mg/L. The authors also noted that the risk of developing severe events is increased by 5% for every one-unit increase in CRP concentration in patients with COVID-19 .
Also in a study it was observed that patients with low oxygen saturation (SpO2?=?90%) had significantly higher levels of CRP (median 76.5?mg/L) compared with patients with high oxygen saturation (SpO2?>?90%) indicating that more severe patients with lung damage have elevated levels of CRP. So, higher levels of CRP indicate more severe disease course-linked to lung injury and worse prognosis .
The CRP concentration decreases when patients inflammatory stimulus ends and the patient is healing.
Table 21: C-Reactive Protein (CRP)

Before After Difference
Drug 71.3 34.7 -36.6
Placebo 24.1 39.6 15.5

Drug Arm:
Mean CRP was 71.3 at day 0 which reduced by 36.6 and became 34.7 on Day 30.
Placebo Arm:
Mean CRP was 24.1 at day 0 which increased by 15.5 and became 39.6 on Day 30.
There is a significant reduction in CRP levels of the drug group as compared to the increase in the CRP level of the placebo group. This indicates that drug is better able to control the inflammation.

2. D Dimer:
D-dimer levels significantly increased with increasing severity of COVID-19 as determined by clinical staging and chest CT staging. Median D-dimer level in non-survivors was significantly higher than in survivors. D-dimer levels correlate with disease severity and are a reliable prognostic marker for in-hospital mortality in patients admitted for COVID-19 .
Higher D-dimer levels were associated with a greater probability of pulmonary embolism
The trial drug has reduced D dimer levels whereas in the placebo group there is increase in D dimer levels which indicates that the placebo group is moving towards severe pathology of COVID 19.
Table 22: D Dimer
D-Dimer Before After Difference
Drug 630 591.05 -38.95
Placebo 534 598 64

Drug Arm:
Mean D Dimer was 630 at day 0 which reduced by 38.95 and became 591.05 on Day 4.
Placebo Arm:
Mean D Dimer was 534 at day 0 which increased by 64 and became 598 on Day 4.
Here too, reduction in Drug arm is prominent compared to slight increase in placebo arm.

3. Sr. Ferritin:
Ferritin is a key mediator of immune dysregulation, especially under extreme hyperferritinemia, via direct immune-suppressive and pro-inflammatory effects, contributing to the cytokine storm A study revealed that in patients who died by COVID-19, ferritin levels were high upon hospital admission and throughout the hospital stay. The median values of serum ferritin levels after day 16 of hospitalization exceeded the upper limit of detection in these patients, suggesting that ferritin levels increased non-stop . There was only sporadic a case for Sr. Ferritin to analyse. Hence it might not be a good idea to mention the number here, as it might be misleading.

Serious Adverse Event:
One SAE was observed during the trial in Placebo group.
SAE: Prolonged hospitalization, Diagnosis: Covid 19 infection related Pneumonia,
Outcome: resolved, Causality: Not related to IP as patient was from Placebo Group
Safety Profile:
Liver Function Tests (LFT) and Renal Function Tests were not deranged at 30 days. Thus, confirming the safety profile of Cap. IP
Conclusion:
The study drug, Cap. IP does seem to have a good potential in curbing down the SARS CoV2 infection.
One could confidently say that the clinical improvement we witnessed in the drug arm must be due to significant decline in the Viral Load and also due to drug’s efficacy in normalising the inflammatory biomarkers.
Cap. IP does seem to arrest the viral replication and it seem to also have the property to pacify the ‘cytokine storm’. The clinical improvement could be due to aggregatory effect of both these actions.

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,CLAIMS:
We claim
1. A first herbo-mineral composition for treating an infection comprising a combination of three herbal medicines and at least one mineral.
2. The herbo-mineral composition as claimed in claim 1 for treating infection of SARS-Cov-2.
3. The herbo-mineral composition as claimed in claim 1 wherein dose of each Herbal Medicine is reduced to 1% - 50 % of its regular dose.
4. The herbo-mineral composition as claimed in claim 1 wherein dose of each Herbal Medicine is reduced to 1% - 25 % of its regular dose.
5. The herbo-mineral composition as claimed in claim 1 wherein the composition comprises herbal medicines Zingiber officinale, Embelia ribes and Glycyrrhiza glabra; and mineral is selected from Calcium, zinc, or combination thereof.
6. The herbomineral composition as claimed in claim 1 wherein the herbal medicines are in the form of Ghana or extract and the mineral is in incinerated form or bhasma form.
7. A second herbo-mineral composition for treating an infection comprising a combination of six herbal medicines and at least one mineral.
8. The herbo-mineral composition as claimed in claim 7 for treating an infection of SARS-Cov-2.
9. The herbo-mineral composition as claimed in claim 7 wherein dose of each Herbal Medicine is reduced to 1% - 50 % of its regular dose.
10. The herbo-mineral composition as claimed in claim 7 wherein dose of each Herbal Medicine is reduced to 1% - 25 % of its regular dose.
11. The herbo-mineral composition as claimed in claim 7 wherein the composition comprises herbal medicines Withania somnifera, Terminalia chebula, Tinospora cordifolia, Emblica officinalis, Asparagus racemosus and Piper longum; and a mineral selected from Calcium, zinc, or combination thereof.
12. The herbomineral composition as claimed in claim 7 wherein the herbal medicines are in the form of Ghana or extract and the mineral is in incinerated form or bhasma form.
13. A kit characterized by having the first Herbo-mineral composition of claim 1 and the second Herbo minereal composition of claim 7.
14. The kit as claimed in claim 13 for treating an infection of SARS-Cov-2.
15. The kit as claimed in claim 13 having from 2 – 20 doses of the first Herbo-mineral composition and from 2 -20 doses of the second Herbo minereal composition.
16. The first herbo-mineral composition of the kit as claimed in claim 11 comprising herbal medicines Zingiber officinale, Embelia ribes and Glycyrrhiza glabra; and a mineral selected from Calcium, zinc, or combination thereof.
17. The second herbo-mineral composition of the kit as claimed in claim 11 comprising herbal medicines Withania somnifera, Terminalia chebula, Tinospora cordifolia, Emblica officinalis, Asparagus racemosus and Piper longum; and a mineral selected from Calcium, zinc, or combination thereof.
18. The herbomineral compositions of kit as claimed in claim 13 wherein the herbal medicines are in the form of Ghana or extract and the mineral is in incinerated form or bhasma form.
19. The first herbomineral composition in any of the preceding claims having
Zingiber officinale from 60 – 120 mg, Embelia ribes from 75 -125 mg, and Glycyrrhiza glabra from 60 – 120 mg; zinc from 20 – 60 mg and calcium from 100 -150 mg.

20. The second herbomineral composition in any of the preceding claims having
Withania somnifera from 100 – 150 mg, Terminalia chebula from 100 – 150 mg, Tinospora cordifolia from 60 -120 mg, Emblica officinalis from 60 -120 mg, Asparagus racemosus 40 - 80 mg and Piper longum 0.05 -20 mg; and zinc from 20 – 60 mg.

21. The first or second herbomineral composition according to any of the preceding claim in the form of a tablet, capsule, caplets, coated tablets, multi-layered tablets, minitablets, pill, powder, granules, seeds, pellet, extrudates, spheronized material, compacts, particulate preparation, dispersion, dispersible tablet, fast disintegrating tablet, lozenge, sublingual or buccal tablets, powder mixture, syrup, suspension, powder for oral solution, powder for oral suspension injectables, liquid, solution, subcutaneous, intravenous, intramuscular composition.

Dated this 2nd day of January 2022

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Ms. Kharkar Pallavi Shashikant
Patent Agent Registration No.: IN/PA-1138 (Agent for Applicant)