Chromatographic and Tandem mass spectroscopic characterization of Ayush Kwath Churnam-an Ayurvedic immunomodulatory drug during COVID-19


 Background: Severe acute respiratory syndrome coronavirus-2, the causative agent of coronavirus disease 2019 (COVID-19) is the global health crisis. There are no existing solutions to COVID 19 as yet. Ayush Kwath Churnam (AKC) is an Ayurvedic medicine promoted by Ministry of AYUSH, Government of India for the enhancement of body immunity in Covid-19 scenario and promoted many AYUSH clinics all over the country with an aim of enhancing immunity of the people to resist SARS Cov-2.Methods: In this study, chemical evaluation of AKC has been done by High Performance Thin Layer Chromatography (HPTLC) and Liquid Chromatography Mass Spectroscopic (LC-MS) analysis. Chemical characterization of AKC was carried out by Electro Spray chemical Ionization (ESI) in both positive and negative mode using Quadrupole Time-of-Flight (Q-TOF) mass spectroscopy. Results: Data processing of molecular ions obtained by mass fragmentation led to the identification of several phytoconstituents belonging to various classes of compounds such as alkaloids, phenolics, flavonoids and isoflavones. Conclusion: The study concluded that AKC contains variety of phytochemicals with numerous biological properties like immunomodulatory, anti-inflammatory and antioxidant activities.


Introduction
Coronavirus disease 2019 (COVID- 19), was declared a pandemic by the World Health Organization and is a major global health concern. How to treat patients with COVID-19 effectively is a common question for health professionals worldwide. To date, therapeutic options for severe COVID-19 remain limited. Severe disease has been associated with changes in peripheral immune activity, including increased levels of pro-in ammatory cytokines that may be produced by a subset of in ammatory monocytes, lymphopenia and T-cell exhaustion [1,2]. Immunomodulatory therapy to down-regulate the cytokine storm may provide insights into the treatment of COVID-19. Natural compounds have contributed enormously to immunomodulatory therapeutics since ancient times. There are thousands of natural compounds that are known to in uence the immune system by either affecting the functions of immune cells or affecting antibody secretion to control the infection and to maintain immune homeostasis. Therefore, it is essential to study the ingredient analysis of herbal preparations to discover novel promising candidates that can be used in the immunotherapeutic strategies for management of Covid-19.
Ayurveda is one of the oldest systems of medicine and the herbal drugs used in the system possess various ethnopharmacological activities such as immunostimulation, neurostimulation, anti-ageing, antibacterial, antiviral, antirheumatic, anticancer, adaptogenic, etc. Ayurveda classics have explained "Rasayana" drugs to enhance body resistance. They consist of a number of plants reputed to promote physical and mental health, improve defense mechanisms of the body and enhance longevity [3,4]. These attributes are similar to the modern concept of adaptogenic agents, which are known to provide protection of the human physiological system against diverse stressors. Many Ayurvedic drugs have been claimed to possess immunomodulatory activity [5][6][7].
Majority of Ayurvedic formulations contain multiple botanicals as ingredient materials, some may be having minerals, metals, and ingredients of animal origin and each of these comprises a number of chemical compounds that may give the anticipated activity in combination. Polyherbal formulations show high effectiveness due to the presence of active phytochemicals which are further potentiated with synergetic interaction of active components of ingredient plants. AKC is prepared out of speci ed parts of four important medicinal plants such as Ocimum tenui orum L., Cinnamomum verum J.Presl, Zingiber o cinale Roscoe and Piper nigrum L. The ingredient analysis of a Polyherbal preparation is very imperative to gain a better understanding of the chemical basis of the therapeutic potential. The present study is focused on chemical analysis of AKC using chromatographic and tandem mass spectroscopic investigations.

Sample Preparation
Ayush Kwath Churnam (AKC) obtained from Product Development Department, Arya Vaidya Sala, Kottakkal, Kerala, India has been taken for the study. 2g of AKC was sonicated with LC/MS grade methanol in an ultra sound bath for 20 minutes. It was then ltered through membrane lter (0.45 µm) and kept under refrigerator until HPTLC and LC/MS analysis.    The mass fragmentation patterns of these compounds are found to be in agreement with that of previous reports [9,15]. Alkaloids such as Piperine (m/z 286.1576) and piperanine (m/z 288.1687) has been reported previously from Piper nigrum [16,17] and are known for their immunomodulatory properties [18,19].

Discussion
Various traditional medicines have been found to modulate various components of innate and acquired immune system. The use of herbal products as immunomodulators has a long history and the ancient times treatment of many diseases were based on modulating the immune system using herbal medicines [20]. The main target of the immunomodulatory natural products is primarily there action on the macrophages which play a key role in the generation of an immune response. The therapeutic potential of immunomodulatory compounds from herbal products and the Ayurvedic concepts of preventive health care have been reported earlier [21]. The present scenario of Covid-19 pandemic also indicates that natural products may be lead compounds, for the development and the discovery of new therapeutic agents for the management of Covid-19.
Ayush Kwath Churnam is recommended by Ministry of AYUSH, Government of India for immunity enhancement in the present situation of pandemic Covid-19. The metabolite pro ling of AKC depicted presence of 27 identi ed phytochemicals belonging to different class of compounds such as alkaloids, phenolics, avonoids and phenolic glycosides. These compounds are reported to possess numerous biological activities including immunomodulatory, anti-in ammatory and anti-oxidant properties. Most of the compounds identi ed from AKC are polyphenolics in nature. Various studies have shown the immunomodulatory effects of polyphenols through different mechanisms such as reformation of composition and level of immunoglobulins, populations of immune cells [22,23]. Many studies have been reported regarding the utility of natural products as immunomodulatory agents to enhance host responses and to reduce damage to the human body. Immunomodulatory effect of caffeic, ferulic, and pcoumaric acids has been reported previously [24]. ESI negative mode ionization showed presence of many compounds from Z. o cinale including 6-Gingerol, 8-shogaol, 8-Gingerdione and 10-gingerdione which have been reported to possess immunomodulatory preporties [25,26]. In ESI positive ionization, alkaloids such as Piperine and Piperanine were detected from AKC and that might have extracted from the ingredient plant Piper nigrum. The immunomodulatory potential of piperine has been reported earlier [27]. Piperine was reported to inhibit proliferative response induced by lipopolysaccharide (LPS) and immunoglobulin α-IgM antibody and resulted in inhibition of IgM antibody secretion and reduced expression of cluster of differentiation CD8612 [28].

Conclusion
The current COVID-19 pandemic is a global unprecedented challenge for human health and researchers are desperately seeking a proven cure for it. When the conventional drugs such as lopinavir, ritonavir, chloroquine, and hydroxychloroquine are not as effective as anticipated, research on traditional herbal medicine is a viable strategy for Covid-19 management and that should be scienti cally validated. The present study concluded that the Ayush Kwath Churna, a popular Ayurvedic drug premeditated during Covid-19 contains many active molecules with known therapeutic potentials like immunomodulatory, anti-in ammatory and anti-oxidant activities and it will be bene cial to acquire immunity against the severe conditions due to the Coronavirus-2 attack.

Declarations
Competing interests: The authors declare no competing interests.
Funding: This work was supported by Navajbhai Ratan Tata Trust, Mumbai, India.