WO2022123605A1 - Herbal composition for covid 19 treatment - Google Patents

Abstract

This invention discloses a mouth dissolving tablet composition comprising at least Piper longum Linn, Piper Nigrum Linn, Zingiber officinale Rosc; and Rock Salt (Saindhav in Sanskrit language) as a composition and also for treatment of one or more of the diseases selected from the group consisting of: (a) Covid-19 for overcoming viral infection, or (b) fungal disease for overcoming fungal infection, or (c) both. The claimed composition is effective for immunomodulation and prevention/prophylaxis from infection of pathogenic microorganisms attaching to ACE2 receptors for initiating an infection, including, but not limited to, infection from SARS-CoV-2 and its mutants. In one aspect this invention comprises an inhaler for prevention/prophylaxis from infection of pathogenic microorganisms attaching to ACE2 receptors for initiating an infection. The pathogenic viruses includes SARS-CoV-2. The inhaler uses ACE 2 inhibitors are essential oils and oleoresins.

Description

HERBAL COMPOSITION FOR COVID 19 TREATMENT

FIELD OF INVENTION

This invention relates to composition for treatment of viral diseases. More particularly, the invention pertains to efficacy of Ardrakadi Nishthivan (Bharat Bhaishajya Ratnakar (BBR) Jvaradhikarl43-145) against COVID-19; and their dosage forms including, without limitation, tablet, capsule, powder, pastilles, inhaler, mouthwash and the like.

BACKGROUND OF THE INVENTION

World is confronting an extraordinary pandemic of COVID 19 caused by Severe Acute Respiratory Syndrome Corona virus 2 (SARS-CoV-2), spread globally with more than 35 million confirmed cases and 1 million deaths till October 2020. This contagion is still continuing to spread and there is no confirmed / assured cure seen coming. The world badly needs clinically-proven prophylaxis and therapeutic strategies. Various systems of Medicine are trying to find solution in some or the other way.

It is acknowledged by WHO (World Health Organization) that no currently available Allopathic medicines, including anti-virals, are effective against COVID-19.

The coronavirus disease (COVID-19) is caused by a virus, NOT by bacteria

The virus that causes COVID-19 is in a family of viruses called Coronaviridae. Antibiotics are not effective against viruses.

Some people who become ill with COVID-19 can also develop a bacterial infection as a complication. In this case, antibiotics may be recommended by a health care provider. There is currently no medication to cure COVID-19. If you have symptoms, call your health care provider or COVID-19 hotline for assistance. Guidance given by World Health Organization is available on the link: https://www.who.int/emergencies/diseases/novel- coronavirus-2019/advice-for public/mythbusters#:~:text=There%20is%20currently%20no%201icensed,19%20hotline%20f or%20assistance

While the work reported here is progressing, current status of knowledge, as on 3 ^rd December 2020is that there is , on treatment of COVID-19, there is no allopathic antiviral that has proven efficacy against COVID- 19 in human beings. In a latest development it has been shown by Cox et (2020) that a therapeutically administered ribonucleoside analogue MK-4482/EIDD-2801 blocks SARS-CoV-2 transmission in ferrets. This is at an early stage of development, has not shown efficacy in human beings; and will take a long time to reach human beings for treatment.

Agarwal et al (2020) have reported that in a clinical trial conducted by Indian Council of Medical Research, Convalescent Plasma therapy has also failed to show any significant impact in treating the COVID-19 patients; and antivirals (Hydroxychloroquine, Remdesivir, Lopinavir/Ritonavir, Oseltamivir), broad spectrum antibiotics are also considered as not effective.

Hence, exploring herbal medicines becomes relevant. Remedies based on Chinese traditional herbs have been investigated in clinical trials and reviewed in a publication available on the link https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7331568/

Xionga et al. (2020) reported eighteen randomized controlled trials (RCTs) in which 2275 patients were enrolled. Most of CHMs were originated from classical Chinese herbal formulas. Liquorice Root (Gancao, Radix glycyrrhizae), Baical Skullcap Root (Huangqin, Radix Scutellaria baicalensis), Pineilia Rhizome (Banxia, Rhizoma pinelliae tematae), Forsythia Fruit (Lianqiao, Fructus Forsythiae suspensae), and Bitter Apricot Seed (Kuxingren, Semen armeniacae amarum) were most frequently used Chinese herbs. The most commonly used dosage formulation was decoction. Their meta-analyses found that comparing CHM group and conventional western medicine group, CHM group has improvements in several clinical parameters including lung CT, clinical cure rate, ranging from mild to critical cases, length of hospital stay, total score of clinical symptoms, fever reduction time, symptom score of fever, number of cough reduction cases, symptom score of cough, number of fatigue reduction cases, symptom score of fatigue, disappearing time of fatigue, TCM syndrome, viral nucleic acid testing, and inflammatory biomarkers (C-reactive protein).

However, the reviewers regarded that firstly, poor methodological design is a very common problem in most of included trials. Significant drawbacks regarding sequence generation of randomization, concealment of allocation, reporting on blinding, dropouts, and pre-estimation of sample size was not done. Secondly, as viral nucleic acid testing turning positive again is very common in the recovery stage of COVID-19, no trial adopted long-term follow-up. Ayurveda is the ancient school of medicine in India which has history of medicines derived from medicinal plants validated for thousands of years for several diseases.

National Clinical Management Protocol based on Ayurveda and Yoga for management of Covid-19 published by Ministry of AYUSH (Ayurveda, Yoga, Unanni, Siddha and Homeopathic medicine) published on date 6^th October, 2020 on (1) Knowledge from Ayurveda classics and experience from clinical practices (2) Empirical evidences and Biological plausibility (3) Emerging trends of ongoing clinical studies. This consensus document is developed by expert committees from All India Institute of Ayurveda (AIIA), Delhi, Institute of Post Graduate Training and Research in Ayurved (IPGTRA), Jamnagar, and National Institute of Ayurveda (NIA), Jaipur, Central Council for Research in Ayurveda (CCRAS), Central Council for Research in Yoga and Naturopathy (CCRYN), other national research organizations. This protocol is for management of mild COVID-19. Moderate to Severe COVID-19 individuals may have informed choice of treatment options. All severe cases will be referred. This protocol and its annexure are approved by the Chairman, Interdisciplinary Committee for inclusion of Ayurveda and Yoga in the management of mild COVID-19 and approved by the empowered committee of the Interdisciplinary AYUSH Research and Development Taskforce on COVID-19, both constituted by the Ministry of AYUSH. Thus, this consensus document represents all that is considered applicable from Ayurveda by a person skilled in the art as well as by those who are highly knowledgeable and qualified in Ayurveda. A complete document entitled “National Clinical Management Protocol based on Ayurveda and Yoga for management of Covid- 19” is available on the link https://www.ayush.gov.in/docs/ayush-Protocol-covid-19.pdf Important highlights of the same are provided below: "General and Physical measures:

1. Gargle with warm water added with a pinch of turmeric and salt. Water boiled with Triphala (dried fruits of Emblica officinalis, Terminalia chebula,Terminalia bellerica') or Yashtimadhu (Glycyrrhiza glabra) also can be used for gargling.

2. Nasal instillation/application of medicated oil (Anutaila or ShadbinduTaila) or plain oil (Sesame or Coconut) or nasal application of cow's ghee (Goghrita) once or twice in a day, especially before going out and after coming back to home.

3. Steam inhalation with Ajwain (Trachyspermum ammi) or Pudina (Mentha spicata) or Eucalyptus oil once a day.

Dietary measures:

1. Use warm water or boiled with herbs like ginger (Zingiber officinale) or coriander (Coriandrum sativum) or basil (Ocimum sanctum / Ocimum basilicum), or cumin (Cuminum cyminum) seeds etc., for drinking purpose.

2. Drink Golden Milk (Half tea spoon Haldi (Curcuma longa) powder in 150 ml hot milk) once at night. Avoid in case of indigestion.

3. Drink AyushKadha or Kwath (hot infusion or decoction) once a day. This Kadha, in 100gmcontains Tulsi (Ocimum sanctum) leaves 44.3gm, Dalchini (Cinnamomum zeylanicum) stem bark 22.3gm, Sunthi (Zingiber officinale) 22.3gm, Krishna Marich (Piper nigrum) 11.1 gm. Less than 1 teaspoon (3 gms) is used to make decoction or herbal tea, consumed once or twice a day by adding the powder to water, adding jaggary to taste and/or Fresh lemon Juice and or resins to taste if needed, the liquid is boiled and strained using a tea strainer/filter. Specific Measures / Symptom Management:

1. Prophylactic care (high risk population, primary contacts): Ashwagandha (Aqueous extract of Withania somnifera Dunal.LinnIP) or its powder: a. 500 mg extract or 1-3 g powder twice daily with warm water for 15 days or one month or as directed by Ayurveda physician. b. Guduchi Ghana vati (Samshamanivati or Giloy Ghana vati having Aqueous extract of Tinospora cordifolia IP) or the powder of Tinospora cordifolia:500 mg extract or 1-3 g powder twice daily with warm water for 15 days or one month or as directed by Ayurveda physician. c. Chyawanaprasha: 10 g with warm water / milk once a day.

2. Asymptomatic COVID-19 positive: a. For prevention of disease progression to symptomatic and severe form and to improve recovery rate: i. Guduchi Ghana vati (Samshamanivati or Giloyvati having Aqueous extract of Tinospora cordifolia (Willd.) Miers.) or the powder of Tinospora cordifolia (Willd.) Miers.: 500 mg extract or 1-3 g powder twice daily with warm water for 15 days or one month or as directed by Ayurveda physician ii. Guduchi + Pippali (Aqueous extracts Tinospora cordifolia (Willd.) Miers. IP and Piper longum L.375 mg twice daily with warm water for 15 days or as directed by Ayurveda physician. iii. AYUSH64: 400 mg twice daily with warm water for 15 days or as directed by Ayurveda physician. 3. Mild COVID-19 Positive: a. Symptomatic management Fever, Headache, Tiredness Dry Cough, Sore throat Nasal congestion: Without evidence of breathlessness or hypoxia

(normal situation): i. Guduchi + Pippali (Aqueous extracts Tinospora cordifolia Wild and Piper longum L.) - 375 mg twice daily with warm water for

15 days or as directed by Ayurveda physician. ii. AYUSH 64: 500 mg twice daily with warm water for 15 days or as directed by Ayurveda physician.

However, it is underlined in this protocol that the interventions and measures proposed in the

Clinical management protocol are not for cure, but for the management of asymptomatic and mild cases of COVID 19 and for prophylactic care. One should not get a false feeling of safety from adopting these measures.

Gudetiet al (2020) have shown that AYUSH-64 along-with standard care in ILI is safe and efficacious and this may use in other viral infections with pyrexia as add-on to standard care for early recovery and better outcome. Each capsule of AYUSH 64 consists of Alstoniascholaris R. Br.(Saptaparna) Bark Aqueous Extract 100 mg., Picrorhizakurroa Royle ex. Benth (Katuki) Root Aqueous Extract 100 mg., Swertiachirata Pexbex. Karst (Kiratatikta) Whole-plant Aqueous Extract 100 mg., Caesalpinia crista L.(Kuberaksha) Seed powder 200 mg.

Wanjarkhedkar et al. reported that ayurvedic formulation was administered as an add-on to

Standard of Care (SoC) in patients with mild to moderate symptoms, in this prospective, open- label, comparative study. Control group received SoC only. Patients receiving Dasamoolkaduthrayam Kashaya (Sahasrayogam) and Guluchyadi kwatham in tablet (Ashtang Hridayam) form in addition to the SoC showed a faster recovery from breathlessness with reduced ageusia . Patients on the treatment group could be discharged earlier than those from the control group. Addition of Dasamoolkaduthrayam kashaya (Sahasrayogam) and Guluchyadi kwatham (Ashtanghridayam)to SoC appeared to accelerate recovery of patients hospitalized for CO VID 19 infection, in terms of reduction of symptoms and duration of hospital stay.

Above two formulations have following contents:

Table 1: Detailed formulation of Dashamoolakadutrayadi Kashaya Gulika tablet

Table 2 Detailed formulation of GuluchyadiKwatham tablet

Early clinical improvement in breathlessness was observed with the present add-on Ayurveda regimen. Ageusia reduced early with add-on Ayurveda regimen. The median duration of hospital stay was reduced; this factor is of importance in view of shortage of hospital beds in

India. The learning from this study is the potential of Ayurvedic therapy in treating COVID 19.

Median hospital stay for COVID pharyngitis patients in the study group was 5 days as compared to 7 days in the control group. The patients with COVID pneumonia had the median hospital stay of 7 days in study group as compared to 8 days in control group.

Thus, here also the Ayurvedic treatment was “an add-on to Standard of Care (SoC)”, and not a first line treatment.

Joshi and Puthiyedath (2020) reported a case study wherein for the first time, the outcomes of

Ayurvedic intervention in a COVID-19 patient with severe hypoxia requiring supportive oxygen therapy. Patient developed fever, severe cough, loss of smell, loss of taste, nasal block, anorexia, headache, body ache, chills, and fatigue and was hospitalized when she developed severe breathing difficulty. Later, she tested positive for COVID-19 by RT PCR. The patient sought Ayurvedic treatment voluntarily when her SPO2 remained at 80% even after being given oxygen support.

The patient was administered Ayurvedic medicines while undergoing oxygen therapy at the hospital. The patient refused to take Fabiflu recommended by the treating physician and discontinued other allopathic drugs except for Vitamin C.

The patient showed clinical improvement within a day of administration of Ayurvedic medicines and was able to talk, eat, and sit on the bed without breathing difficulty and her SPO2 became stable between 95 and 98%. In the next two days, she was asymptomatic without oxygen support and was discharged from the hospital in the following week. Since obesity and high plasma CRP indicated high risk for progression to severe disease, the favorable outcomes with Ayurvedic treatment in this patient is significant and warrants further studies.

Ayurveda care may be considered as first -line cost-effective alternative for COVID-19 patients presenting with symptomatic hypoxia in an integrative setup.

She was administered the following Ayurvedic medicines immediately.

1. Sadangapaniyam with Guduci: One sip - every 10 minutes for 3 days, One sip every half hour for next 2 days, One sip every two hours for next 6 days.

2. Saddharanacurna: 1 pinch every hour for 7 days. 3. Suksmatriphala: 250 mg tab at bed time for four days

4. Kanakasavam: 1 ml every two hours for first three days and 1 ml four times a day for next 8 days

4. Indukantam Kasayam: 1 ml every two hours for first three days, 1 ml four times a day for next 8 days.

On first day of hospitalization she was prescribed: Azee (Azithromycin) 625 mg 1-0-0, Cefexime 200 mg 1-0-1, T. Dexamethasone 6 mg 1-0-0, T. Acetaminophen 650 mg SOS, T. Vitamin C 500 mg 1-0-1, T. Pantoprazole 40 mg 1-0-0, C. Becosule (Vitamin B Complex) 1- 0-0, Syp. Grilinctus (Guaifenesin, Chlorpheniramine maleate and Ammonium chloride) 2 tsp 1-1-1. In addition, she was prescribed by the doctors the option of starting Fabiflu, but the patient declined to take this medicine. She stopped taking all the above prescribed medicines after starting the Ayurvedic treatment (22 June 2020 by 7 pm). Only Vitamin C and oxygen support was continued. By 24.6.2020 her cough became mild and breathing difficulty was experienced only on exertion. All other symptoms subsided except for mild headache. The SPO2 levels improved and stabilised at 95-98%. She was maintained on intermittent oxygen, which was completely withdrawn on 26 June 2020 by 7 pm. Futher, Ayurvedic treatment was continued along with Vitamin C supplementation. She was not given any other medications except on the first two days of hospitalization prior to starting Ayurvedic treatment.

This is first report of Ayurvedic treatment being efficacious without Allopathic treatment except for vitamin C. However, this protocol of Ayrvedic treatment included five formulations and very elaborate frequency of administration of the medicines, which is very difficult for compliance. Hence, there was still a need for lesser number of formulations/ingredients and simple schedule of administration of the medicines.

SUMMARY OF THE INVENTION

This invention discloses a composition comprising at least Piper longum Linn, Piper Nigrum Linn, Zingiber officinale Rose; and Rock Salt for treatment of patients of one or more of the diseases selected from the group consisting of: (a) Covid- 19 for overcoming viral infection, or (b) fungal disease for overcoming fungal infection, or (c) both.

This invention also discloses a composition comprising at least Piper longum Linn, Piper Nigrum Linn, Zingiber officinale Rose, Rock Salt and ACE2 inhibitor active ingredients of herbs.

In one embodiment the composition as disclosed in above two paragraphs is a mouth dissolving tablet.

The composition is efficacious on the fungal infections including, but not limited to, infection from Mucor sp., Aspergillus sp.and Candida sp.

The composition the fungal infection comprises infection from one or more fungi including, but not limited to, Rhizopus caespitosus, Rhizopus delemari, Rhizopus homothallicus, Rhizopus microspores, Rhizopus oryzae, Rhizopus reflexus, Rhizopus schipperae, Rhizopus stolonifer (black bread mold), Rhizopus arrhizus, Rhizopus oligosporus, , Rhizopus circinans, Rhizopus lyococcus, Rhizopus americanus, Rhizopus azygosporus, Rhizopus rhizopodiformis, Rhizopus niveus and Rhizopus sexuahs; Mucor amphibiorum, Mucor circinelloides, Mucor ellipsoideus, Mucor fragilis, Mucor hiemali, Mucor hiemalis f. silvaticus, Mucor. indicus, Mucor mucedo, Mucor paronychius, Mucor piriformis, Mucor phimbeus, Mucor pseudolusitanicus, Mucor racemosus, Mucor ramosissimus, Mucor variicolumellatus, Mucor vehutinosus, Apophysomyces variabilis, Apophysomyces trapeziformis, Apophysomyces ossiformis: Lichtheimia ramosa L. corymbifera, Cunninghamella binarieae R.Y. Zheng 2001, Cunninghamella blakesleeana, Cunninghamella Candida Yosh. Yamam. 1929, Cunninghamella clavata R.Y. Zheng & G.Q.Chen 1998, Cunninghamella echinulata (Thaxt.) Thaxt. ex Blakeslee 1905, Cunninghamella elegans Lendn. 1905, Cunninghamella homothallica Komin. & Tubaki 1952, Cunninghamella intermedia K.B.Deshp. & Mantri 1966, Cunninghamella multiverticillata R.Y. Zheng & G.Q. Chen 2001, Cunninghamella phaeospora Boedijn 1959, Cunninghamella polymorpha Pispek 1929, Cunninghamella septata R.Y.Zheng 2001 and Cunninghamella vesiculosa P.C.Misra 1966, Aspergillus flavus , Aspergillus fumigatus, Candida albicans and Candida glabrata.

The inventive composition as claimed herein is effective for immunomodulation and prevention/prophylaxis from infection of pathogenic microorganisms attaching to ACE2 receptors for initiating an infection, including, but not limited to, infection from SARS-CoV-2 and its mutants.

The ACE2 inhibitors used as ingredient in the compositions of this invention include one or more, without limitation, extracts from Nicotiana benthamiana, Pelargonium graveolens L’Hér Momordica dioica roxb.ex willd, Valeriana jatamansijones ex roxb., Oroxylum indicum (L.), Artemisia absinthium L, Syzygium aromaticum (L.), Phaseolus vulgaris L Inula helenium L, Allium sativum L, Piper longum L, Ipomoea obscura (L.), Ipomoea obscura (L.), Piper nigrum L, Piper retrofractum vahl, Melaleuca cajuputi maton, Scutellaria baicalensis georgi, Erigeron breviscapus, Glycyrrhiza glabra L, Glycyrrhiza uralensis fisch. ex DC. Bupleurum spp. Heteromorpha spp., Scrophularia scorodonia L, Withania somnifera (L.), Asparagus racemosus willd., Diplocyclos palmatus (L.), Citrus spp., Vitis vinifera L. and Aframomum melegueta K.Schum

In one aspect this invention comprises an inhaler for immunomodulation and prevention/prophylaxis from infection of pathogenic microorganisms attaching to ACE2 receptors for initiating an infection. The pathogenic viruses include SARS-CoV-2.

The inhaler uses ACE 2 inhbitors are essential oils and oleoresins. The essential oils include, one or more, without limitation, Wintergreen Oil, Nilgiri/Eucalyptus oil, Terpeon Oil/Terpene oil, Camphor, Lemongrass oil, Terpinene, Anethole, Camphene, Carvacrol, Caryophyllene, Cinnamaldehyde, Cinnamyl acetate, Citral, Citronella, Citronellol, Criminal, Estragole, Eucalyptol, Eugenol, Fenchel, Geraniol, Umonene, Linalool, Menthol, Myrtanol, Ocimene, p- Cymene, Pinocarveol, Pulegone, Sabinene, Sylvestrene, Terpinen-4-ol, Thujene, Thymol and Zingiberene. The ACE2 inhibitors include, without limitation, one or more of zingiberene, zingiberenece, Gingerol, Eugenol, A co run calamus L, Zingiber officinale Rose, Trachyspermum ammi, Trachyspermum ammi L. and Syzygium aromaticum L & L.M., Nicotiana benthamiana, Pelargonium graveolens L’Hér. Momordica dioica roxb.ex willd, Valeriana jatamansijones ex roxb., Oroxylum indicum (L.), Artemisia absinthium L, Syzygium aromaticum (L.), Phaseolus vulgaris L Inula helenium L, Allium sativum L, Piper longum L, Ipomoea obscura (L.), Ipomoea obscura (L.), Piper nigrum L, Piper retrofractum vahl, Melaleuca cajuputi maton, Scutellaria baicalensis georgi, Erigeron breviscapus, Glycyrrhiza glabra L, Glycyrrhiza uralensis fisch. ex DC. Bupleurum spp. Heteromorpha spp., Scrophularia scorodonia L, Withania somnifera (L.), Asparagus racemosus willd., Diplocyclos palmatus (L.), Citrus spp., Vitis vinifera L. and Aframomum melegueta K. Schum

DETAILED DESCRIPTION OF THE INVENTION

“National Clinical Management Protocol based on Ayurveda and Yoga for management of Covid- 19” published by Ministry of AYUSH, Government of India, is an authentic source of current status of Ayurveda in the context of Covid- 19 treatment. It has very categorically concluded with clear advice that:

“ - the interventions and measures proposed in the Clinical management protocol are not for cure, but for the management of asymptomatic and mild cases of COVID 19 and for prophylactic care. One should not get a false feeling of safety from adopting these measures”

On this background, it was surprising to find one Ayurvedic formulation, known in Ayurveda scriptures as “Ardrakadi Nishthivan” in which worked with very high virucidal efficacy as stand-alone treatment in absence of Standard-care-of-Allopathic Covid- 19 treatment; and has provided a complete control on infection, a complete cure to Covid-19 patients within a matter of 72 hours with administration of a tablet each four times a day.

Subsequently, this formulation has also been added with ingredients that would make it useful as prophylactic too.

The “Ardrakadi Nishthivan" is described in the ancient reference BHARAT BHAISHAJYA RATNAKAR (BBR)(Jvaradhikarl43 to 146^rdverse) as a powder. It is one of the embodiments of this invention that we have made the powder into a mouth disintegrating tablet. Details of Ardrakadi Nishthivan reference in BBR are as follows:

(English translation of above verses-

• Composition -Take equal quantity of Rock salt, Ginger, long pepper and black pepper. Whole formulation processed with fresh juice of ginger.

• Dosage administration - Put that powder in mouth some time and spit it out. Do repeatedly. • Therapeutic effect - This composition will help to cure Fever Fainting Dry or wet cough, Pain in throat Overall drowsiness, heaviness in body , Hyperacidity , Indigestion , Kapha in heart

• Translation of the indication provided in the Verse 143 is as follows

Table 3

• These indications are of Influenza and Pneumonia, which are not Covid-19. It is now well known that treatments that are known to cure Influenza and Pneumonia do not cure Covid-19. The Covid-19 symptoms which are not shared in above Verse 143 are: Anosmia, Rhinitis, Anxiety, Breathlessness, diarrhea, loss of taste or smell a rash on skin, or discoloration of fingers or toes in mild cases, Lung fibrosis, difficulty breathing or shortness of breath, chest pain or pressure, and loss of speech or movement. Hence, the Verse 143 by no means suggests that this formulation shall cure Covid-19.

• This is perhaps the reason that even after about 9 months of Cobid- 19 pandemic in India and about 12 months in China, no practitioner in Ayurveda, including the Ministry of Ayush nor above cited publications using Ayurvedic formulations ever contemplated that a single Ayurvedic tablet formulation in general and Ardrakadi Nishthivan in particular given four times a day shall provide an efficacious and a virucidal medication for Covid-19 providing cure for the patient in three days; where all chemical/Allopathic virucides have failed.

• Thus, it is an embodiment of this invention that the Ayurvedic formulation Ardrakadi Nishthivan which is converted into mouth dissolving tablet, rather than as powder given in original reference, has been seen surprisingly, not just as an immunity booster but as a highly efficacious virucide against SARS-CoV 2 and as a remedy for Covid- 19 that has better efficacy than the State-of-Art virucides. This has not been anticipated by publication in any document in the context of Covid-19 nor used for this purpose in the country, there is no known state-of-art medicine on Covid-19; this is a breakthrough in treatment of the dreaded pandemic of Covid-19. Hence, this is a case of totally unanticipated and totally unobvious new use, a breakthrough for Ardrakadi Nishthivan, and not just a mere discovery of new use for known substance. This new use has not fallen in public domain nor is part of State of Art treatment anywhere in the world for Covid- 19

The Medicinal properties of its ingredients are given as follows

Pippali (Piper longum Linn)-

• This herb is known from the ancient Sanskrit Ayurvedic texts. It is believed that Pippali was first mentioned in “Charaka Samhita” (IV-II B.C.) - the ancient Indian treatise to a healthy and balanced way of living. Very often it has been described as a remedy for the treatment of respiratory diseases, as well as for the treatment of the problems related to the intestinal flora. In the past it was even used to treat diseases such as cholera, tuberculosis, tetanus and leprosy.

• According to the classification of Ayurveda, it is heavy (to digest), slightly oily and it has moisturizing properties. The long pepper is very powerful and it has a quick and almost immediate effect after consumption.

Black Piper (Piper nigrum Linn)

• Black pepper, (Piper nigrum Linn), also called pepper, perennial climbing vine of the family Piperaceae and the hotly pungent spice made from its fruits. Black pepper is native to the Malabar Coast of India and is one of the earliest spices known. Widely used as a spice around the world, pepper also has a limited usage in medicine as a carminative (to relieve flatulence) and as a stimulant of gastric secretions.

Sunthi (Zingiber officinale Rose.)

• Zingiber officinale Rose., commonly known as ginger, is a spice consumed worldwide for culinary and medicinal purposes. The plant has a number of chemicals responsible for its medicinal properties, such as anti-arthritis, anti- inflammatory, antidiabetic, antibacterial, antifungal, anticancer etc. The present chapter compiled scientific data retrieved from websites, such as PubMed, Science Direct, Scopus, Web-of-Knowledge, Google Scholar, and others related to the phytochemistry and pharmacology of ginger. A synopsis of the world production of the plant as well as some patents related to Z. officinale are also provided.

Rock salt (Halite; ‘Saindhav’ in Sanskrit language)

• It's a superior salt, according to Ayurveda and is a highly crystalline salt.

• Rock salt, on the other hand, is already found in solid form and is simply mined. This type of salt is also known as halite and often comes in larger crystals or has a coarser texture.

Fresh juice of ginger

Improves digestion. Is helpful in cough, cold; and relieves congestion and pain. It also controls high blood pressure, and has antiviral properties Pharmacodynamics of Ardrakadi Nishthivan tablets

Following are general principles applicable for Ayurvedic therapy; which are applicable to Ardrakadi Nishthivan tablets also.

1. Ayurvedic ingredients work in synergism and deliver efficacy as a whole in combination with each other.

2. Single herb has hundreds of phyto -ingredients and in poly-herbal formulation there is further heterogeneity in phyto -ingredients. Ayurveda is not a single molecule therapy so BA, BE or Pharmacodynamics of a poly-herbal composition is always a challenge. Answer to this is found in Reverse pharmacology.

3. All Phyto -ingredient molecules work in synergy. If one tries to Isolate single molecule or take aqueous / alcoholic extract, it may fail to deliver expected result and may even have side effects.

4. Use herb as a whole is always safe and has optimum therapeutic effects.

This invention also discloses immunomodulator formulations. Immunomodulators are medications used to help regulate or normalize the immune system. Examples include one class of immunomodulation which is used as an add-on therapy to treat support in viral, bacterial and fungal infections and even in auto immune disorders.

Literature references for ingredients as immunomodulator is provided in Table 4.

Table 4: Immune Boosting structures of herbs and properties of the ingredients of

Immunity Booster formulation

Above all references mentioned in table no. 4 are known for their individual immune booster or therapeutic or both activities

The immunomodulator formulation was made in the instant invention comprising the ingredients provided in Table 5; the efficacies of which are also mentioned in the Table 5. Table 5

Ingredients and their respective efficacy

An invention is claimed also in an inhaler for prevention and prophylaxis from infection of pathogenic microorganisms attaching to ACE2 receptors for initiating an infection. In one embodiment of the invention, the inhaler is made from oils and oleoresins of herbs having

ACE2 blocking activity receptor blocker activity (Dear DR. Milind: Please confirm again by making reference work on whether the phrase “having ACE2 blocking activity receptor blocker activity” is applicable to oils as well as the oleoresins of hers. Clarity and accuracy on this is very much essential from the point of view of claims to be made. Is it possible that “Oils” serve as “carriers” for the oleoresins and the oleoreins are only actives which are ACE inhibitors? For oils please check where is their mention made as ACE inhibitors and cite the same here) and not as ACE inhibitors . In another embodiment, the inhaler made from oleoresins of herbs having ACE2 receptor blocker activity is used as prophylactic/preventive for infection from Coronavirus- 19 infection. It is also an embodiment of this invention wherein inhalers of oleoresins of selected herbs having ACE2 receptor blocker activity are used to keep a person healthy. It is also an embodiment of this invention wherein inhalers the oleoresins include, without limitations, zingiberene, zingiberenece, Gingerol and Eugenol.

Yadalam et al (2021) have listed following essential oils as anti-virals against SARS-CoV-2 which can be used in inhaler: Terpinene, Anethole, Camphene, Carvacrol, Caryophyllene, Cinnamaldehyde, Cinnamyl acetate, Citral, Citronella, Citronellol, Cuminal, Estragole, Eucalyptol, Eugenol, Fenchel, Geraniol, Umonene, Linalool, Menthol, Myrtanol, Ocimene, p- Cymene, Pinocarveol, Pulegone, Sabinene, Sylvestrene, Terpinen-4-ol, Thujene, Thymol and Zingiberene.

Muchtaridi et al (2020) have enlisted Plant derivatives with potential effect on the SpikeRBD/TMPRSS2/ACE2 axis. The list comprises: Nicotiana benthamiana, Pelargonium graveolens L’Hér Momordica dioica roxb.ex willd, Valeriana jatamansijones ex roxb., Oroxylum indicum (L.), Artemisia absinthium L, Syzygium aromaticum (L.), Phaseolus vulgaris L Inula helenium L, Allium sativum L, Piper longum L, Ipomoea obscura (L.), Ipomoea obscura (L.), Piper nigrum L, Piper retrofractum vahl, Melaleuca cajuputi maton, Scutellaria baicalensis georgi, Erigeron breviscapus, Glycyrrhiza glabra L, Glycyrrhiza uralensis fisch. ex DC. Bupleurum spp. Heteromorpha spp., Scrophularia scorodonia L, Withania somnifera (L.), Asparagus racemosus willd., Diplocyclos palmatus (L.), Citrus spp., Vitis vinifera L. and Aframomum melegueta K.Schum. A person skilled in the art immediately understands that oleoresins form all these plants can be sued in the inhaler according to this invention.

Thus. In addition to case studies on Covid- 19 patients, in non -clinical in-vitro studies, Ardrakadi Nishthivan is shown in Example 6 to have antiviral activity against non- enveloped virus, in enveloped virus as well as having anti-fungal activity. These observations explain the observed surprising effect of Ardrakadi Nishthivan in below mentioned case studies on Covid-19 patients and in clinical trial reported below. The anti- fungal properties are also very much relevant for Covid-19 patients since almost an epidemic of fungal diseases have been recorded post-recovery from Covid- 19 that includes Mucormycosis, Candidosis and Aspergillosis. These fungal diseases are killer diseases and are expensive to treat also. A person skilled in the art can certainly make out from the above examples that use of Ardrakadi Nishthivan in main treatment will not allow the fungal diseases also to come up at all; and in other therapies where Ardrakadi Nishthivan was not used and the fungal diseases that have occurred concurrent to or post-Covid- 19 can potentially be treated using Ardrakadi Nishthivan. The link https://www.cdc.gov/fungal/diseases/mucormycosis/definition.html provides information that Mucormycosis is caused by

Rhizopus species, Mucor species, Rhizomucor species, Syncephalastrum species, Cunningha mella bertholletiae, Apophysomyces species, and Lichtheimia (formerly Absidia) species. Example 6 has shown that Ardrakari Nishthivan has excellent efficacy against Mucor racemosus ATCC 42647. A person skilled in the art would understand that Ardrakari Nishthivan would be efficacious against all fungi that cause Mucromycosis. The fungi that cause Mucormycosis includes Rhizopus caespitosus, Rhizopus delemari, Rhizopus homothallicus, Rhizopus microspores, Rhizopus oryzae, Rhizopus reflexus, Rhizopus schipperae, Rhizopus stolonifer (black bread mold), Rhizopus arrhizus, Rhizopus oligosporus, , Rhizopus circinans, Rhizopus lyococcus, Rhizopus americanus, Rhizopus azygosporus, Rhizopus rhizopodiformis, Rhizopus niveus and Rhizopus sexualis; Mucor amphibiorum, Mucor circinelloides, Mucor ellipsoideus, Mucor fragilis, Mucor hiemali, Mucor hiemalis f. silvaticus, Mucor. indicus, Mucor mucedo, Mucor paronychius, Mucor piriformis, Mucor phimbeus, Mucor pseudohisitanicus, Mucor racemosus, Mucor ramosissimus, Mucor variicolumellatus, Mucor vehitinosus, Apophysomyces variabilis, Apophysomyces trapeziformis, Apophysomyces ossiformis: Lichtheimia ramosa L. corymbifera, Cunninghamella binarieae R.Y. Zheng 2001, Cunninghamella blakesleeana, Cunninghamella Candida Yosh.Yamam. 1929, Cunninghamella clavata R.Y. Zheng & G.Q.Chen 1998, Cunninghamella echinulata (Thaxt.) Thaxt. ex Blakeslee 1905, Cunninghamella elegans Lendn. 1905, Cunninghamella homothallica Komin. & Tubaki 1952, Cunninghamella intermedia K.B.Deshp. & Mantri 1966, Cunninghamella multiverticillata R.Y. Zheng & G.Q. Chen 2001, Cunninghamella phaeospora Boedijn 1959, Cunninghamella polymorpha Pispek 1929, Cunninghamella septata R.Y. Zheng 2001 and Cunninghamella vesiculosa P.C.Misra 1966

Muchtarid et al (2020) have mentioned that Aspergillus flavus, Aspergillus fumigatus cause A spergillom icosis. Abdullah et al. (2021) have mentioned that Candida albicans and Candida glabrata are causal fungi of Candidosis in Covid-19 patients.

Since Ardrakari Nishthivan has been shown to be effective against the pahtogenic fungus

Mucor recemosus, and the fmgicieds have generic efficacy against all fingi,a person skille din the art can recognise that it shall be equally efficacious against above list fo fingi also which cause Mucormycosis, Aspergillosis and Candidosis.

Results provided in Table 8 clearly indicate following properties of the Investigational

Product (mouth dissolving tablets of Ardrakadi Nishthivan):

1. Antiviral as therapeutic effect 2. Prophylactic as ACE2 receptor blocker effect

3. Inducer of salivation, providing locally viral wash effect

4. Reduction in the risk of further viral infections and secondary bacterial infections colonization.

5. Efficacious dosage: 1 mouth dissolving tablet of Ardrakadi Nishthivan 3 to 4 times at regular intervals.

EXAMPLES

EXAMPLE 1

Ardrakadi Nishthivan its ingredients and method of preparation Verse 143 describes ingredients and composition of Ardrakadi Nishthivan original reference from BHARAT BHAISHAJYA RATNAKAR. This powder contains following ingredients: Piper longum Linn, Piper Nigrum Linn, Zingiber officinale Rose; and Rock Salt. Table 6

Method of preparation specified in BBR is as follows:

1. T ake all ingredients numbered from 1 to 4

2. Dry those in shade and then pulverize to powder. 3. Mix the powders well in grinder.

4. Crush fresh ginger after thorough wash with water.

5. Put the paste in a clean cotton cloth. 6. Squeeze the juice out.

7. Add sufficient ginger juice to the powder-mix in mortar and pestle such that the powder-mix should be completely immersed in the ginger juice.

8. Start the grinding till all the powder gets soaked in the juice well

9. Grind for 1 hour

10. Let it dry completely

11. make the tablets.

Example 2

In April 2020, a patient was diagnosed with COVID 19. He was home quarantined. He was treated with the allopathic medicines. After treatment also he was not feeling fresh and mild fever continued. Given standard care of treatment was the one which was recommended at that time for CO VID 19, the same was as follows:

• Azithromycin 500 OD 3 days

• VitaminC500 mg+Zink 10 mg+ Multivitamin especially B12 daily

• Paracetamol 650 TDS SOS

• In first 7 days Tab Favipiravir

After telephonic conversation with him, he was treated with Ayurvedic ODT (Orally disintegrating tablet) of “Ardrakadi Nishthivan

In the instant example, which was carried out while there was a lockdown and access to laboratories and ready-made Ardrakadi Nishthivan was not available, the same was prepared by collecting all ingredients and as per the process described in the treatise BBR. Pills were prepared at home and the dose advised was 1 pill (by allowing dissolution in mouth) three times a day. Surprisingly, all symptoms of the patient were gone completely in 4 days.

Example 3

Efficacy of Ardrakadi Nishthivan tablets

Case I - A middle aged female patient of 45 years was suffering from Covid-19 symptoms. Dry cough, mild fever always below 99⁰Fahrenheit, occasional sneezing and continuous sore. She was treated immediately after detection of symptoms. She was not receiving any State-of- art treatment. Since she was of close acquaintance to me, and she knew that I am an Ayurvedic medical practitioner, she desired that I should give her an Ayurvedic treatment. Hence, we tried giving her Ardrakadi Nishthivan as sole treatment by making the tablets at home. To our surprise, she got relief within 3 days from date of giving mouth dissolving tablets of Ardrakadi Nishthivan for 4 times in a day.

PCR test for diagnosis of Covid-19

The COVID-19 RT-PCR test is a well-known real-time reverse transcription polymerase chain reaction (RT-PCR) test for the qualitative detection of nucleic acid of the causal virus SARS- CoV-2 in upper and lower respiratory specimens (such as nasopharyngeal or oropharyngeal swabs, sputum, lower respiratory tract aspirates, bronchoalveolar lavage, and nasopharyngeal wash/aspirate) collected from individuals suspected of patients suspected of COVID-19 by their healthcare provider (HCP); as well as upper respiratory specimens (such as nasopharyngeal or oropharyngeal swabs, nasal swabs, or mid-turbinate swabs) collected from any individual, including for testing of individuals without symptoms or other reasons to suspect COVID19 infection

(https://www.fda.gov/media/136151/download)These tests were used in the examples described here. Example 4

Documented Case Studies

It was decided to check viral load i.e., quantitative PCR through Indian Council of Medical Research (ICMR) recognised laboratory (address: GenePath DX, Above Phadake Hospital, 1206/B Jungli Maharaj Road Shivajinagar Pune 411004 www.genepathdx.com) initially in 2 patients of COVID-19 by treating them with Ardrakadi Nishthivan mouth dissolving tablets.

The observations are as follows:

Table no. 7

Surprising decline in virus load in one hour was observed. After these initial observations, we decided to go for the pilot study of 4 patients.

Table 8: Pilot study done on 4 patients . 500 mg 1 mouth dissolving tablets of Ardrakadi Nishthivan 4 hourly for 3 days at regular intervals between 8 am to 8 pm .Nasal+Oral swab taken for quantitative Viral load (RTqPCR) each 24 hrly.

Decline in 99% of the viral load is considered as virus free status of the patient. Since there was no treatment between 72 and 96 hours, the patients are considered to have become virus free with maximum 72 hours of treatment. Example 5

IMMUNOMODULATOR FORMULATION

Immunity Booster Formulation comprise following ingredients”

Table 9: Immunity Booster Formulation

Process of making above formulation comprised following steps:

1. Aqueous extract of ingredients 1 to 5 that were readily available in market were procured. In the alternative, the extracts can also be made by methods well known to a person of an ordinary skill in the art. 2. Ingredient 6 was also procured from the market and the same is readily available.

3. Ingredient 7 is prepared by sohxlet extraction process using solvent ether, hexane using seeds of Zanthoxylum armatum DC.

4. Ingredients 1 to 7 were mixed together as per the percentage provided above, and the mixture was prepared. 5. Fresh juice of ginger was prepared and added in mixture.

6. Whole mixture is dried and powdered. This powder is the API (Active Pharmaceutical Ingredient) of the Immunity Booster Formulation.

This API can be converted in tablet, Orally Dissolving Tablet, syrup, lozenges, capsules, Pills, pastilles as per standard industrial dosage forms by methods well known in the art.

Example 6

NON-CLINICAL IN VITRO ANTI-VIRAL EFFICACY STUDIES

1. Ardrakadi Nishthivan

Name of the test - Assess the Activity of Microbicides against Viruses in Suspension

Test Method: - ASTM E 1052: 2020

Scope of Method: This test determines if test product can inactivate virus in suspension. One part of the virus suspension with nine Parts of the test substance are held at the desired temperature for the required contact time and then assayed for viable virus in an appropriate host system.

Experimental Conditions:

Experimental Conditions:

Test Product : 10 mg/ml Cone. Contact time : 4 hours

Organic Load : 0.5gpl BSA Bovine serum albumin

Test Method : 24 Well plate, Neutralisation and Dilution method

Neutralization : SCDLP medium Soyabean Casein Digest Medium Medium of Cell culture : Eagle’s minimal essential medium (EMEM), supplemented with inactivated FBS Foetal Bovine serum & antibiotics

Virus strains and host cells: Virus strains and hosA12:D17t cells:

Test Virus: Human Coronavirus HCoV-229E (Surrogate of SARS-CoV-2)

Host Cell: MRC-5 cell line (ATCC CCL-171); Passage No.: Cells from PN 16 Infectivity titre test: TCID50 method Results:

Test Virus: Human Coronavirus HCoV-229E (Surrogate of SARS-CoV-2)

Results:

Test Virus: Human Coronavirus HCoV-229E (Surrogate of SARS-CoV-2)

1. Test Sample: Thinqure 20 (Trademark for Ardrakadi Nishthivan) 10 mg/ml Cone.

Table 10

Note:

HCoV-229E belongs to the family Coronaviridae and subfamily Orthocoronavirinae.

HCoV-229E is one of seven known coronaviruses that infect humans. The other six are Human HCoV-OC43, HCoV-NL63, HCoV- HKU1, MERS-CoV, SARS-CoV-1, SARS-CoV-2.

The standardized preparation of Ardrakadi Nishthivan was provided by the inventors for these studies and analyses were got done by outsourcing analytical services from BIOTECH TESTING SERVICES (7O4|LO5, Malwa, Patanwalalnd. Estate, L.B.S. Marg, Ghatkopar (W), Mumbai - 400086, INDIA) (A) Activity of Ardrakadi Nishthivan against Viruses in Suspension

Table 11

Sample Description

Test Method:

ASTM E 1052: 2020

Scope of Method:

This test determines if test product can inactivate virus in suspension. One part of the virus suspension with nine Parts of the test substance are held at the desired temperature for the required contact time and then assayed for viable virus in an appropriate host system.

Experimental Conditions: Ardrakadi Nishthivan : 1mg/ml, 10 mg/ml and 50 mg/ml The above test sample of Ardrakadi Nishthivan was prepared by following procedure:

1. Ardrakari Nishthivan formulation 50 g was added to 500 ml of solvent

2. The sample was refluxed in Soxhlet for 6 hours

3. Two different solvents were used and carried out in two different Soxhlet apparatus i.e. Ethanol and Methanol

4. The extracts were concentrated in rotary evaporator

5. The concentrate was dried in hot air oven

6. Both the extracts were diluted in 1 :2 volume in distilled water in 0.1%

Phosphate Buffer Saline to give combined concentration of 100 mg/ml

7. The samples were further diluted in respective media for specific tests at either

50mg/ml, 10mg/ml and 1mg/ml

8. The extracts were stored at 4°C.

Contact time : 1 hours and 4 hours

Organic Load : 0.5gpl BSA

Test Method : 24 Well plate, Neutralisation and Dilution method

Neutralization : Soybean Casein Lecithin Polysorbate (SCDLP) medium

Medium of Cell culture: Eagle’s minimal essential medium (EMEM), supplemented with inactivated FBS (Fetal Bovine Serum) and antibiotics Virus strains and host cells:

Test Virus: Influenza A virus (H3N2): A/Hong Kong/8/68: ATCC VR-1679

Host Cell: MDCK cell ATCC CCL-34

Infectivity titre test: TCID50 method Test Procedure:

1. The prepared Virus inoculum is added to the test product and the virus recovery control media at a ratio of 1 part virus + 9 parts test product.

2. At the end of contact time, the test and recovery suspensions are neutralized.

3. An aliquot removed from the cytotoxicity control and inoculated with a low- concentration viral suspension was used to generate the neutralization control to confirm the efficacy of neutralization method.

4. The neutralized test, recovery, cytotoxicity control, and neutralization control suspensions were serially diluted in the appropriate media. Each dilution is plated in quadruplicate to host cell monolayers in a 24-well plate. 5. Plates were incubated for 5 - 7 days duration for Visible CPE (cytopathic effect by examined under microscope and Confirmatory TCID 50 method.

Results:

Test Virus: Influenza A Virus (H3N2): A/Hong Kong/8/68: ATCC VR-1679 Table 12

1. Test Sample: Ardrakadi Nishthivan — 1mg/ml

Test Virus: Influenza A Virus (H3N2): A/Hong Kong/8/68: ATCC VR-1679

Table 13

Test Sample: Ardrakadi Nishthivan — 1mg/ml

Results:

Test Virus: Influenza A Virus (H3N2): A/Hong Kong/8/68: ATCC VR-1679

12

Table 14

2. Test Sample: Ardrakari Nishthivan — 10 mg/ml

Test Virus: Influenza A Virus (H3N2): A/Hong Kong/8/68: ATCC VR-1679

Table 15 Test Samplc: Ardrakadi Nishthivan— 10 mg/ml

Results:

Test Virus: Influenza A Virus (H3N2): A/Hong Kong/8/68: ATCC VR-1679

Table 16

3. Test Sample: Ardrakadi Nishthivan — 50mg/ml

Test Virus: Influenza A Virus (H3N2): A/Hong Kong/8/68: ATCC VR-1679

Table 17

Test Sample: Ardrakadi Nishthivan — 50 mg/ml

(B) Activity of Ardrakadi Nishthivan against Viruses in Suspension

Table 18

SAMPLE DESCRIPTION : Test sample labeled as:THINQURE20 (Trade Name of Ardrakadi Nishthivan formulation)

Test Method:

ASTM E 1052: 2020

Scope of Method: This test determines if test product can inactivate virus in suspension. One part of the virus suspension with nine parts of the test substance are held at the desired temperature for the required contact time and then assayed for viable virus in an appropriate host system.

Experimental Details: Test product concentration : Ready to use

Virus : MS2Bacteriophage;

Permissive Host Cell : Escherichia coli ATCC15597

Interfering agent : No soiling agent was used

Contact Period : 1 hour and 4 hours

Neutralizer : DE broth Medium : Trypticase soya agar

Incubation for survivors : 37°C for 3 days

Validation and Records:

Table 19

Neutralizer Validation and Records:

Where -

A=No. of PFU/ml of Test organism in

Experimental condition validation B=No. of PFU/ml of Test organism in

Neutralizer Toxicity validation

Test Procedure:

Thoroughly mixed virus suspension was added to nine parts of Test substrate. It was held at Room temperature for 1 minute and 10 minutes following exposure time, aliquots were withdrawn, neutralized and by serial tenfold dilution assayed to determined surviving Viruses in comparison with Control without test product. Virus assay was quantitative as Plaque forming unit (PFU) visible as area of Clearance. Results:

Table 20

INTERPRETATION:

Test product Ardrakadi Nishthivan formulation — 10 mg/ml has shown 66.66% and 54.76% reduction; Ardrakadi Nishthivan formulation — 50 mg/ml hasShown78.92%and85.00%reduction of MS2 Bacteriophage as surrogate virus in

1 hour and 4 hours when tested as per standard ASTM E 1052: 2020. (C) Time Kill Test

SAMPLE DESCRIPTION : Test sample labeled as: Ardrakadi Nishthivan

Test Standard:

ASTM E 2315 - 16

Test Inoculum:

Mucor racemosus ATCC 42647

Test Conditions:

Concentration 1mg/ml, 10mg/ml and 50mg/ml

Diluent/Neutraliser Lecithin, Polysorbate80, Sodium thiosulphate, Histidine, Saponinin

Phosphate buffer 0.0025mol/l

Contact time : 1 hours and 4 hours

Contact Temperature : Room temperature

Media and Reagent : Sabourauds Dextrose agar, incubated at 28°C

Procedure:

Product was inoculated with test fungus individually (approximately10⁸CFU/ml). After the specified exposure time of 1 hour and 4 hours surviving microorganisms were recovered by drawing an aliquot, neutralizing and performing Standard Pour plate Technique. Culture count was ascertained by dilution Blank. Adequate Validation of Neutralizing agent was also carried. Test was carried out in duplicate and average count was taken as CFU/ml.

Neutralizer Validation: Table 21

Results:

Table 22

Percentage Reduction of Microorganism = 100(Initial — After Exposure) / Initial Log Reduction = Log Initial - Log After Exposure

INTERPRETATION:

Sample labeled as Ardrakadi Nishthivan — 1mg /ml has shown >1 log reduction I 92% reduction; Ardrakadi Nishthivan- lOmg/ml has shown >1 log reduction / 93% reduction; Ardrakadi Nishthivan— 50mg/ml has shown >1 log reduction/94% reduction of test fungus viz. Mucor racemosus in 1 hour and 4 hours when analysed as per ASTME 2315 — 16 Method.

In summary:

• Antiviral activity against Human Coronavirus HC₀V-229E - Ardrakadi Nishthivan was tested at 10 mg/ml for anti-Corona virus activity on cell line MRC-5 and has shown 97% kill against Coronavirus (ASTM E 1052: 2020) in 4 hrs.

• Antiviral activity against Non-Enveloped virus -Ardrakadi Nishthivan — 50 mg/ml has shown 85.00% reduction of MS2 Bacteriophage as surrogate virus in 4 hours per standard ASTM E 1052: 2020

• Antiviral activity against Enveloped virus - Ardrakadi Nishthivan — 50 mg/ml has shown 99.00% reduction of INFLUENZA as surrogate virus in 4 hours ASTM E 1052: 2020 • Antifungal activity- Arar akadi Nishthivan 50mg/ml has shown reduction 96% reduction of test fungus ASTM E 2315 - 16 Method.

Example 7

CTRI registered Clinical Trial Report of Ardrakadi Nishthivan

STUDY TITLE

A Non-Interventional, Retrospective, Observational study to analyze Safety, Efficacy and Tolerability of Ardrakadi Nishthivan in COVID-19 patients.

For these studies, standardized A rdrakadi Nishthivan was made available by the inventors and clinical trial services were sourced from Mediclin Clinical research 4^th Floor Ambika Industries, Opp Thakur Mall, Mira Road Thane 401107

MATERIALS AND METHODS

Materials

The Thiqure 20 - A standardized and ready to use formulation comprising Ardrakari Nishthivan was obtained from Thinq pharma CRO, Mumbai.

Standardization of Ardrakadi Nishthivan

The Ardrakadi Nishthivan was standardized by HPLC method to evaluate the piperine and gingerol content in the formulation.

Study design and objectives

This was a non-interventional, retrospective, observational study conducted between 12-Aug- 2020 and 12-Nov-2020. The primary objective of the study was to evaluate efficacy of Ardrakadi Nishthivan in Covid-19 patients. The secondary objectives were to analyze safety, and tolerability of the formulation following fixed dose combination treatment with time in Covid patients. The study was performed in Yashwantrao Chavan Memorial Hospital, Pune, India. The study was performed in compliance with good clinical practice and all applicable laws and regulations. The necessary approvals for study design were approved by Ethicare Ethics Committee and the protocol was registered with Clinical Trial Registry of India (CTRI) with Registration No.: CTRI/2021/03/032471. All procedures followed the tenets of the Declaration of Helsinki, were in accordance with all regulatory standards, were approved by an Institutional Review Board and all subjects signed an informed consent form.

Patients

The study included male (21) and female (09) patients between agel8 to 75 years with diagnosis of Covid positive by RT -qPCR test. For inclusion in the study, patient must have had RT-PCR test positive for both oral and nasal swabs. The patients who were diagnosed Covid positive and were taking Ardrakadi Nishthivan as prescribed by physician were also included in the study. The patients with history of intracranial bleeding, incomplete medical history, or suffering from haemoglobinopathies, active malignancies were excluded from the study. The pregnant or lactating women, smokers, alcohol drinkers, and the females not ready to use acceptable contraceptive methods during treatment periods were also excluded from the study. After screening with inclusion and exclusion criteria, total 30patients were included in the study. At the time of screening, a written informed consent was taken from all the subjects and their demographics, complete medical history, past history and history of any other treatment was recorded. Additionally, vital signs including pulse, BP, temperature, body weight, and physical examination and Viral load by RT-qPCR were also assessed. Treatment

The patients were asked to take tablets in the mouth and slowly to be sucked (licked within mouth by tongue) till entire tablet is dissolved in mouth and not to swallow or chew or crush. The first dose was 2 tablets of Ardrakadi Nishthivan and from second dose it was reduced to 1 tablet at a time at 4 hours interval; thus on day 1, five tablets were given. From day 2 to day 5 one tablet was given every 4 hrs. (08:00 AM, 12:00 noon, 04:00 PM, and 08:00 PM). Out of 30 patients, 29 completed the treatment, one patient was excluded from the study due to not completing the treatment.

Outcome parameters

The change in viral load from the baseline to end of study was recorded with RT-qPCR test. Evaluation of vital signs including pulse, BP, temperature, body weight, and physical examination, recording of concomitant medication, Dietary and lifestyle counselling were done to evaluate the safety of Ardrakadi Nishthivan formulation (Thinqure20™). The safety and tolerability of the Ardrakadi Nishthivan was determined by recording all serious adverse events (SAE) and adverse events (AE) regardless of treatment group or suspected causal relationship to study drug.

Statistical analysis

The viral load after treatment was statistically compared with viral load before treatment by performing Wilcoxon rank sum test as it is performed when sample size is below 40. The data was represented as Mean ± SD (Standard Deviation). The p< 0.05 was considered as significant. The analysis was performed using statistical software GraphPad Prism Version 8. EFFICACY RESULTS AND TABULATIONS OF INDIVIDUAL SUBJECT DATA

Analysis of Efficacy

Efficacy of the investigational product was analyzed on the basis of increase in the Viral load from baseline to end of study visit. As per the statistical analysis plan, the efficacy data was analyzed for effect on individual and cumulative efficacy parameter.

• Primary Efficacy Parameter

A) Changes in Viral Load:

p-value calculated using chi-square test. The below table is considered for analysis of viral load after log10 transformation:

Viral load

The treatment with Ardrakadi Nishthivan for 5 days significantly (p<0.001) reduced the viral load in the Covid 19 Patients when compared with viral load before treatment (Figure 1).

Statistical analysis

The viral load after treatment was statistically compared with viral load before treatment by performing Wilcoxon rank sum test as it is performed when sample size is below 40. The data was represented as Mean ± SD (Standard Deviation). The p< 0.05 was considered as significant. The analysis was performed using statistical software GraphPad Prism Version 8.

DISCUSSION

The relationship between Covid-19 virus load and the severity of disease progression is poorly characterized in Covid- 19. The plasma viral load is directly associated with progression of disease severity (Fajnzylber et al, 2020; Pujadaset al. 2020). Increase in plasma viral load leads to worsening of disease severity, marked reduction in lymphocytes count, and significant increase in plasma inflammatory markers such as interleukin 6 (IL-6), and C-reactive protein (CRP) leading to increased mortality (Fajnzylber et al, 2020).

Ardrakadi Nishthivan treatment showed significant reduction in the viral load indicating its beneficial role in the inhibition of disease progression and mortality rate. In addition, the active components of the Ardrakadi Nishthivan (Piperine and Gingerol, Zingeberene) possess potent anti-inflammatory and antiarthritic effects of piperin in human interleukin 1 -stimulated fibroblast-like synoviocytes and in rat arthritis models activities (Bang et al. 2009) and Anti- oxidative and anti-inflammatory effects of ginger in health and physical activity (Mashhadi et al. 2013) may also help in reducing the cytokine storm and further worsening of conditions.

The angiotensin converting enzyme - 2 (ACE2) receptors play a very pivotal role in viral entry and its expression in the major vital organs such as heart, kidney and lungs (Habas et al. 2019). SARS-CoV-2 virus causes balance between ACE and ACE2 and activate the renin angiotensin aldosterone system (RAAS) leading to entry of virus in host cell and its replication (Beyerstedt 2021). Inhibition of ACE2 receptors and RAAS system can limit the viral load and disease progression by inhibiting viral entry and replication in the host cells 1. The piperine, zingiberene and gingerol have proven ACE inhibitory activity (Maurya et al, 2020; Bare et al. 2020) This action of Ardrakadi Nishthivan may be attributed to ACE2 inhibition and inhibition of viral entry in the host cells. Founded results suggests that phytochemicals from Ardrakadi Nishthivan might be effective as a therapeutic as well as prophylaxis against COVID-19 by preventing viral attachment to the host cells through inhibition of spike glycoprotein. Molecular docking results are indicated with excellent binding affinity of phytochemical piperine, Zingiberene against SARS-CoV-2 spike glycoprotein and its cellular receptor along with MolDock score and other parameters (Maurya et al, 2020; Bare et al.

Ardrakadi Nishthivan.

Mole dock study shows phytochemicals from of Zingiber officinale as entry inhibitor of SARS- COVID 2 VIRUS. Gingerol and Zingiberene had effective binding activity with ACE2 receptors (Maurya et al, 2020; Bare et al. 2020; Chakotiya and Sharma 2020).

Ardrakadi Nishthivan comprising of Piper longumLinn, Piper nigrumLinn. Zingiber officinale Rose and rock salt, possess various active agents like piperine, Zingiberene, gingerol which play their role for its effectiveness against SARS-Co2 virus. Ardrakadi Nishthivan might be acting on single target or multiple targets for inhibiting SARS-CoV2 viral load.

Covid-19 can cause significant discomfort, continuous cough, fever/high temperature (37.8°C or greater), Loss of, or change in, sense of smell or taste, respiratory failure, shock, or multi- organ dysfunction, reduce quality of life, and lead to a loss of productivity. In this study Ardrakadi Nishthivan have been shown to improve quality of life in Covid-19 patients. Treatment with Ardrakadi Nishthivan was safe and it was well tolerated. Adverse event experienced during clinical trial, all of them were mild in the nature. In addition, there were no serious adverse events reported at any time during the study.

CONCLUSION

Evidence supports that the use of Ardrakadi Nishthivan has beneficial effects in treatment of Covidl9 which may be linked to decrease in total viral load in patients. It provided better relief for recovery of the COVID-19 disease. Ardrakadi Nishthivan is well-tolerated, safe and is effective in COVID-19 patients, especially with reference to the reduction in viral load.

Example 8

Herbal Inhaler having ACE2 receptor blocking activity In the following is an illustrative example of the inhaler made using ACE2 inhibitors.

Table 25

1. Composition

Method of Preparation a. Preparation of base A: Ingredients from 1 to 6 are bought in market and mixed in quantity indicated in Table 25 to prepare base A b. Preparation of base B: Ingredients 7, 8, 9 and 10 were prepared as Oleoresins by solvent extraction in Soxhlet Apparatus and mixed in quantity indicated in Table 25 to prepare base B. i. Vekhand Oil (oil of Acorus calamus L) was prepared by extracting rhizome was extracted by using soxhlet method .solvent used hexane, pet ether ii. Sunthi Oil (Oil ofZinziber officinale Rose) was prepared by extracting rhizome was extracted by using soxhlet method .solvent used hexane, or petroleum ether. iii. Ajwain oil Oil (oil of Trachyspermum ammi (L.) Sprague ex Turrill) was prepared by extracting seeds was extracted by using soxhlet method .solvent used hexane or pet ether iv. Clove oil Oil (oil of Syzygium aromaticum L & L.M. ) flowder budswas extracted by using soxhlet method, solvent used hexane or petroleum ether

1. These extracts were mixed together in quantity indicated in Table 25 to prepare Base B c. Final API (Active Pharma Ingredient) is prepared by mixing Base A with Base B in a mixer accompanied with stirring for 15 minutes. 3. Dosage form a. Standard inhaler - cotton absorbent stick is filled with API as per optimum level.

Dose b. As a prophylaxis for adults - 2 deep inhalations in each nostril 1 hrly c. 2 year to 12 year - 1 inhalation in each nostril once a day. d. Children under 12 years (with adult supervision) 1 inhalation in each nostril once a day. e. Not to use for children below 2 years f. Or as per directed by physician

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Claims

CLAIMS:

1. A composition comprising at least Piper longum Linn, Piper Nigrum Linn, Zingiber officinale Rose; and Rock Salt (in Sanskrit called as 'Saindhav') for treatment of patients of one or more of the diseases selected from the group consisting of: a. Covid-19 for overcoming viral infection, or b. fungal disease for overcoming fungal infection, or c. both.

2. A composition comprising at least Piper longum Linn, Piper Nigrum Linn, Zingiber officinale Rose, Rock Salt and ACE2 inhibitor active ingredients of herbs.

3. The composition as claimed in claim l is a mouth dissolving tablet.

4. The composition as claimed in claim 1 wherein the fungal infection comprises infection from Mucor sp., Aspergillus sp.and Candida sp.

5. The composition as claimed in claim 4 wherein the fungal infection comprises one or more selected from the group consisting of Rhizopus caespitosus, Rhizopus delemari, Rhizopus homothallicus, Rhizopus microspores, Rhizopus oryzae, Rhizopus reflexus, Rhizopus schipperae, Rhizopus stolonifer (black bread mold), Rhizopus arrhizus, Rhizopus oligosporus, , Rhizopus circinans, Rhizopus lyococcus, Rhizopus americanus, Rhizopus azygosporus, Rhizopus rhizopodiformis, Rhizopus niveus and Rhizopus sexualis; Mucor amphibiorum, Mucor circinelloides, Mucor ellipsoideus, Mucor fragilis, Mucor hiemali, Mucor hiemalis f. silvaticus, Mucor. indicus, Mucor mucedo, Mucor paronychius, Mucor piriformis, Mucor plumbeus, Mucor pseudolusitanicus, Mucor racemosus, Mucor ramosissimus, Mucor variicolumellatus, Mucor velutinosus, Apophysomyces variabilis,Apophysomyces trapeziformis, Apophysomyces ossiformis.’Lichtheimia ramosa L. corymbifera, Cunningham ella binarieae R.Y. Zheng 2001, Cunninghamella blakesleeana, Cunninghamella Candida Yosh.Yamam. 1929, Cunninghamella clavata R.Y. Zheng & G.Q.Chen 1998, Cunninghamella echinulata (Thaxt.) Thaxt. ex Blakeslee 1905, Cunninghamella elegans Lendn. 1905, Cunninghamella homothallica Komin. & Tubaki 1952, Cunninghamella intermedia K.B.Deshp. & Mantri 1966, Cunninghamella multiverticillata R.Y.Zheng & G.Q. Chen 2001, Cunninghamella phaeospora Boedijn 1959, Cunninghamella polymorpha Pispek 1929, Cunninghamella septata R.Y. Zheng 2001 and Cunninghamella vesiculosa P.C.Misra 1966, Aspergillus flavus , Aspergillus fumigatus, Candida albicans and Candida glabrata. The composition as claimed in claim 2 for immunomodulation and prevention/prophylaxis from infection of pathogenic microorganisms attaching to ACE2 receptors for initiating an infection. The composition as claimed in claim 6 wherein the infection of pathogenic microorganisms attaching to ACE2 receptors for initiating an infection comprises infection from SARS-CoV-2 and its mutants. The composition as claimed in the claim 2 wherein the ACE2 inhibitors are selected one or more from the group consisting of Nicotiana benthamiana, Pelargonium graveolens L’Hér Momordica dioica roxb.ex willd, Valeriana jatamansijones ex roxb., Oroxylum indicum (L.), Artemisia absinthium L, Syzygium aromaticum (L.), Phaseolus vulgaris L Inula helenium L, Allium sativum L, Piper longum L, Ipomoea obscura (L.), Ipomoea obscura (L.), Piper nigrum L, Piper retrofractum vahl, Melaleuca cajuputi maton, Scutellaria baicalensis georgi, Erigeron breviscapus, Glycyrrhiza glabra L, Glycyrrhiza uralensis fisch. ex DC. Bupleurum spp. Heteromorpha spp., Scrophularia scorodonia L, Withania somnifera (L.), Asparagus racemosus willd., Diplocyclos palmatus (L.), Citrus spp., Vitis vinifera L. Aframomum melegueta K.Schum An inhaler for prevention/prophylaxis from infection of pathogenic microorganisms attaching to ACE2 receptors for initiating an infection. The inhaler as claimed in claim 9 wherein the pathogenic microorganisms attaching to ACE2 receptors for initiating an infection is a Coronavirus. The inhaler as claimed in claim 9 wherein the Coronavirus is SARS-CoV-2. The inhaler as claimed in claim 9 wherein the ACE 2 inhibitors are essential oils and oleoresins. The inhaler as claimed in claim 10 wherein: a. the one or more essential oils are selected from the group consisting of Wintergreen Oil, Nilgiri/Eucalyptus oil, Terpeon Oil/Terpene oil, Camphor, Lemongrass oil, Terpinene, Anethole, Camphene, Carvacrol, Caryophyllene, Cinnamaldehyde, Cinnamyl acetate, Citral, Citronella, Citronellol, Cuminal, Estragole, Eucalyptol, Eugenol, Fenchel, Geraniol, Umonene, Linalool, Menthol, Myrtanol, Ocimene, p-Cymene, Pinocarveol, Pulegone, Sabinene, Sylvestrene, Terpinen-4-ol, Thujene, Thymol and Zingiberene., and. b. the ACE2 inhibitors are selected, one or more, from the group consisting of zingiberene, zingiberenece, Gingerol, Eugenol, Acorun calamus L, Zingiber officinale Rose, Trachyspermum ammi, Trachyspermum ammi L. and Syzygium aromaticum L & L.M., Nicotiana benthamiana, Pelargonium graveolens L’Hér Momordica dioica roxb.ex willd, Valeriana jatamansijones ex roxb., Oroxylum indicum (L.), Artemisia absinthium L, Syzygium aromaticum (L.), Phaseolus vulgaris L Inula helenium L, Allium sativum L, Piper longum L, Ipomoea obscura (L.), Ipomoea obscura (L.), Piper nigrum L, Piper retrofractum vahl, Melaleuca cajuputi maton, Scutellaria baicalensis georgi, Erigeron breviscapus, Glycyrrhiza glabra L, Glycyrrhiza uralensis fisch. ex DC. Bupleurum spp. Heteromorpha spp., Scrophularia scorodonia L, Withania somnifera (L.), Asparagus racemosus willd., Diplocyclos palmatus (L.), Citrus spp., Vitis vinifera L. and Aframomum melegueta K.Schum,