STUDY TITLE:

ANTIOXIDANT POTENTIAL, FREE RAADICAL SCAVENGING ACTIVITY, IMMUNOMODULATION EFFECT, EFFECT ON QUALITY OF SLEEP AND ON STRESS OF WAGH BAKRI PREMIUM SPICE TEA WITH 7 SPICE EXTRACTS (WB-7)- A CLINICAL TRIAL IN HEALTHY VOLUNTEERS.

BACKGROUND:

Recent studies have shown that the Oleoresin of Ginger, Black Pepper, Cardamom, Piper longum, Clove, Cinnamon & Nutmeg afford significant protection against stress. It has several known health benefits in respiratory disorders, diabetes mellitus, skin disorders, pain management and acts also as immune modulators.

Presently, huge numbers of people in developing countries depend on medicinal plants extracts for healthcare, skin care, economic benefits, and cultural development. For centuries, medicinal plants have been widely used in traditional medicine in countries like India, China, Germany, Thailand, etc. [1]. The World Health Organization (WHO) projected that 80% of the population relies on traditional medicine, which is clearly elucidated by the 19.4 billion USD global revenue for herbal remedies in 2010 [2]. Moreover, the demand for traditional medicinal plants is increasing; for instance, the market for medicinal plants is expanding at an annual rate of 20% in India. Likewise, in China, 30% to 50% of the total medicinal consumption is from preparations of traditional medicine [3]. Nearly 76.7% of the citizens of Thailand have reported mainly using traditional herbal extracts for their primary healthcare [4]. Around 90% of the German population uses natural remedies for certain health issues [5]. Therefore, the medicinal plant extracts used in traditional medical treatments are significant in both developing and industrialized countries. This is clearly demonstrated by the worldwide market for traditional medicine. This market continues to gradually increase [6].

The extracts from Oleoresin of Ginger, Black Pepper, Cardamom, Piper longum, Clove, Cinnamon & Nutmeg among the 7 global species belonging to the above extracts, are available in India. They are more important due to its huge spectrum of traditional medicinal properties. For centuries, these plant extracts have been used in diverse forms and holds a place of pride in Indian Ayurvedic medicine. Different parts of, such as its leaves, root, bark, and fruit, are known to promote various biological activities. Aromatic bioactive constituents in the leaves retain their flavor and other qualities, even after drying. On the other hand, limited studies have been conducted for evaluating the details, like phytochemical screening, identification, and pharmacological activities, will be systematically categorized, compared, and summarized. We hypothesized that, through all of these efforts, a good summary on pharmacological activities that could initiate future perspectives with the utmost clarity could be produced.

EFFECT OF SECONDARY METABOLITES FORM DIFFERENT PARTS OF PLANTS

 Antioxidants

Reactive oxygen species (ROS), such as singlet oxygen (O₂), hydrogen peroxide (H₂O₂), the superoxide anion (O₂•^−), and the hydroxyl radical (•OH), are often generated as by products of cellular metabolic reactions and exogenous induction. These ROS create homeostatic imbalances, which lead to the generation of oxidative stress, which in turn, induces cell death and tissue injury [7]. ROS in elevated levels can damage biomolecules such as nucleic acids, proteins, and lipids [8]. Even though the antioxidant defense systems like enzymatic antioxidants and non-enzymatic antioxidants are functioning, uncontrolled ROS accumulation during the life cycle promotes the development of age-dependent diseases, like cancer, atherosclerosis, arthritis etc. [9]. Natural antioxidants from plant sources have been considered a promising therapy for the prevention and treatment of these diseases, especially neurodegenerative disorders, cardiovascular diseases, cancer, and other conditions.

Oxidative Stress

Chemical species with one or more unpaired electrons are called free radicals. In biological systems, the term “free radicals” refers to reactive oxygen species (ROS). Major ROS include O₂•^−, H₂O₂, and •OH [10]. In addition to ROS, reactive nitrogen species (RNS), including peroxynitrite (NO₃^−), NO, and S-nitrosothiols, also contributes to the generation of oxidative stress. Both ROS and RNS arise as intermediates in several metabolic processes and are specifically produced as part of the cellular defense against invasive pathogens. Free radicals also regulate many processes, including cellular growth, glucose metabolism, and proliferation [11].

Apart from certain beneficial effects, free radicals induce various deleterious effects. In a non-specific manner, ROS can react on significant biomolecules, which leads to deleterious effects like a loss of enzyme activity, genetic mutations and permeability alterations in the cell membrane, and RNS-induced protein S-nitrosylation [12]. Because DNA is constantly attacked by the free radicals, around 75,000 to 100,000 DNA damage events might occur in each cell per day. •OH is the most reactive species and interacts with all biological molecules, including the C-8 position of guanine to form 8-hydroxyguanine, which is one of the most frequently found oxidized bases in DNA [13]. An increase in the free radical concentration in the body can cause subsequent oxidative and cellular damage to lipids, proteins, RNA, and DNA [14].

Mitochondrial Dysfunction

Mitochondria are the primary source of high-energy metabolism within the cell. Mitochondria are known as the powerhouse of the living cell. Mitochondria also regulate calcium homeostasis and play a role in scavenging free radicals and controlling programmed cell death and/or the apoptosis-signaling pathway [15]. Mitochondrial damage leads to reduced adenosine triphosphate (ATP) production, increased ROS generation, impaired calcium buffering, damage to mitochondrial DNA (mtDNA), an altered mitochondrial morphology, and alterations in mitochondrial fission and fusion. All these events eventually lead to cell death [16]. It is currently believed that the majority of ROS are generated by mitochondrial complexes I and III, likely due to the release of electrons by NADH and dihydroflavine-adenine dinucleotide (FADH₂) into the electron transport chain (ETC). Mitochondrial dysfunction is a characteristic of all chronic diseases and aging. It is characterized by a loss of efficiency in the ETC, as well as reductions in the synthesis of high-energy molecules [17]. These diseases include neurodegenerative diseases like Parkinson’s disease (PD), Alzheimer’s disease (AD), amyotrophic lateral sclerosis, multiple sclerosis, Huntington’s disease, cardiovascular diseases, auto-immune diseases, diabetes, and others [18,19].

Mitochondria, as essential organelles, have a noteworthy role in the viability of neuronal cells. Excess ROS formation is due to complex I inhibition inducing impairments in the mitochondrial membrane potential (MMP) and the pro-apoptotic members are believed to permeabilize the outer mitochondrial membrane due to the formation of oligomeric pores, which permits the release of apoptogenic molecules from the intermembrane space. Recent studies have evaluated that neuroprotective activities of isolongifolene and structurally similar compounds, such as girinimbine, murrayazoline, and O-methylmurrayamine A isolated from plant extracts .By using different in vitro assays, a study reported that the above bioactive compounds exhibited the ability to restore the MMP levels and repair the mitochondrial damage [20,21,22].

 Inflammation

Tissue injury, cell damage, infections due to pathogens, and alterations in biochemical lead to a biological response called inflammation. In neurological disorders, the important components involved in inflammatory processes are believed to be mast cells, ependymal cells, microglia, astrocytes, and macrophages [23]. Microglia, a type of neuronal support cell acting as resident macrophages located throughout the brain by changing their morphology, actively respond to inflammation and participate in removing damaged neurons and pathogens. An ethanol extract from leaves showed significant analgesic and anti-inflammatory activity when explored using carrageenan-induced hind paw edema in albino rats [24]. Another study also confirmed the anti-inflammatory activity of a leaf extract in carrageenan-induced paw oedema [25]. Additionally, the study recognized the analgesic activity of curry leaves with several experimental models. Leaf extracts effectively attenuate the pain which is induced by an intraperitoneal injection of acetic acid and subplantar injection of formalin in mice, and the analgesic effect was elucidated with the writhing responses and pain responses in the late phase. Furthermore, it was reported that higher concentrations (20 and 40 mg/kg, per OS (P.O.) reduced the early-phase inflammatory responses induced by formalin [26]. The above studies have shed light on the mechanism of the anti-inflammatory activity of leave extracts and their active compounds, which are comparable to nonsteroidal anti-inflammatory drugs (NSAIDs).

Apoptosis

Apoptosis is a physiological programmed cell death mechanism which is essential for the flawless growth and development of organisms. Furthermore, it is a lively physiological course causing the self-destruction of cells that comprises lethal biochemical and morphological changes in the nucleus and cytoplasm. During cellular strain like oxidative stress and DNA damage, the process of apoptosis can arise, particularly in cells with high proliferation rates and a high expression of pro-apoptotic genes [27]. Intrinsic and extrinsic pathways regulate apoptosis, but both pathways are associated and the molecules involved in those pathways can influence one another. It also inducted apoptosis and cell cycle arrest due to inhibition of the PI3K/AKT/mTOR and Wnt/b-catenin signalling pathways [28].

JUSTIFICATION OF THE STUDY:

Each stage of the body’s immune response relies on the presence of many micronutrients. Examples of nutrients that have been identified as critical for the growth and function of immune cells include vitamin C, vitamin D, zinc, selenium, iron, and protein (including the amino acid glutamine).[i],[ii]. The nutrients present in Masala tea may help the immune system in several ways: working as an antioxidant to protect healthy cells, supporting growth and activity of immune cells, and producing antibodies.

TYPE OF STUDY:   Clinical Trial Single group

AIM:

To assess the total Antioxidant Potential, Lipid Peroxidation activity (MDA) and immunomodulation effect of Wagh Bakri premium spice tea mixture with 7 spice extracts (WB-7) in healthy volunteers.

OBJECTIVE:

1.     To assess the efficiency of Total Antioxidant Potential / (Vit A, Vit E, Vit C) of WB-7 in healthy volunteers on 1^st day, and on 30^th day.

2.     To Study the Lipid peroxidation activity in terms of Serum malondialdehyde level (MDA) a measure of oxidative stress indices of WB-7 in healthy volunteers on 1^st day and on 30^th day.

3.     To evaluate immune response (IgG, IgE, IL6) of WB-7 in healthy volunteers on 1^st day and on 30^th day to ascertain the immune response developed by the Individual.

4.     To assess the subjective parameters like Pittsburg Sleep Quality Index (PSQI) questionnaire and Hamilton Depression Rating Scale (HDRS) to assess day to day stress level & Sleep quality on weekly intervals.

METHODOLOGY:

Study Sites:

Department of Swasthavritta and Yoga, Department of Rasashastra and BK and PSAM Hospital, Biochemistry department SIMSR

Study duration:  6 Months

This study will be prospective single group study to evaluate the immune-modulatory effect of Masala tea with Natural spice Extracts (Oleoresin of Ginger, Black Pepper, Cardamom, Piper longum, Clove, Cinnamon & Nutmeg) in healthy volunteers. The study will be carried out after obtaining clearance from the SIMSR Institutional Ethics Committee (IEC). The study duration is for 6 months in which a volunteer will be observed for a period of 1 months of follow-ups (i.e. 0 Baseline, and at 30 days).

During the study period, 40 healthy volunteers willing to participate will be enrolled and will be provided with WB7 Tea twice a day for 30 days under direct observation once in the morning after breakfast and once in the evening.

The volunteers will be kept on dietary restriction and drug restriction which are already proven to have effect on immune system.

 The volunteers will be evaluated for subjective parameters weekly and objective parameters on base line i.e. on 1^st day and on 30^th day from the period of enrolment.

Blood collection: 10 ml of venous blood will be collected from the Subjects on 1^st day and on 30^th day under conditions of fasting. Blood also collected from the subjects in the control group. The analysis samples will be divided into heparinized tubes, EDTA-containing tubes, and plain tubes without anticoagulant. Plasma-EDTA and serum will be obtained by centrifugation at 1500 g for 10 minutes at 4°C.

 To assess lipid peroxidation the plasma MDA in terms of TBARS will be assessed, Total antioxidant enzymes, blood samples were stored at -20°C until analysis. CBC, ESR, Electrolytes, Proteins, LFT, RFT, Lipid profile, as well as immune response (IgG, IgE, IL6) levels in the serum will be determined immediately after collection.

Inclusion Criteria:   

Patients fulfilling all the following criteria will be included:

1.     Volunteers willing to participate in the study and understand the informed consent document

2.     Relatively poor wellbeing with score of 13 or below based on the WHO (Five) Well-Being Index

3.     Age 20 to 60 years; both males and females

Exclusion Criteria:    

Patients will be excluded with the following reasons:

1.     Patient with any of the systemic disorders

2.     Patients having depression and undergoing treatment for any of psychiatric disorder

3.     Pregnant women.

4.     Patients who are under other investigational drugs within the preceding month.

5.     The volunteers who encounter any disease during study period will be excluded.

STATISTICAL METHODS: All Statistical analysis in the study performed using SPSS for window version 20.0.  The results of laboratory tests in the study to be summarized as a mean ± Standard deviation (SD) (Descriptive statistics), Chi-square test (Categorical variables) and Independent ‘t’ test (comparing with two groups).

OUTCOME MEASURES:

Volunteers may coupe better with day to day stress level and may show improved immunity.

Potential risks and benefits: Cost compatibility, timeline

Ethical considerations and methods to address issues: Ethical clearance from IEC

 BUDGET (GIVE DETAILS) AND PROPOSED FUNDING SOURCE: As a part of sponsored project by Gujarat Tea Processors & Packers Ltd

CONFLICT OF INTEREST: All the investigators are not from Wagh Bakri industry and therefore there is no Conflict of interest, As per the clinical trial agreement with sponsoring body the intellectual property and study outcome belongs to the sponsoring body. However publications can be done after obtaining permission from sponsoring body.

Following biomarkers assessment will be done.