Description:
FORM 2

THE PATENTS ACT, 1970
(Act 39 of 1970)
AND
THE PATENTS RULES, 2003

COMPLETE SPECIFICATION
(See section 10 and rule 13)

TITLE OF THE INVENTION

“FORMULATION FOR MODULATING PAIN AND INFLAMMATION AND METHODS THEREOF”

APPLICANT
Purobien Lifesciences Private Limited of Plot No. 41, Upper Ground Floor, Block-C, Pkt-9, Sec-8, Opp. Saroj Hosp., Rohini Delhi, North West DL 110085,

PREAMBLE TO THE DESCRIPTION
The following specification describes the invention.

FIELD OF INVENTION

The present invention provides a formulation PL02 for modulating (preventive and therapeutic treatment) pain and inflammation related disorders such as osteoarthritis and other joint pain. The present invention further provides methods of using the formulation PL02 for treating pain and inflammation mediated disorders, such as neurological diseases, autoimmune, metabolic disorders, bone and joint diseases.

BACKGROUND OF INVENTION
Inflammation is a protective reaction and part of complex biological response of body/ tissues to any harmful stimuli (internal or external), such as damaged cells, pathogens and irritants.It involving immune cells, blood vessels, and molecular mediators released by cells. Further it can be classified as Acute or Chronic Inflammation. Both acute and chronic inflammation is a generic response, and considered as a mechanism of innate immunity. Too little inflammation could lead to progressive tissue destruction by the harmful stimulus (e.g. pathogen) and compromise the survival of the organism. In contrast, chronic inflammation which can continue for months to years is associated with various diseases, such as diabetes, hay fever, cancer, ulcerative colitis, periodontal disease, atherosclerosis, and osteoarthritis. In contrast acute inflammation is an instant process occurring in response to tissue injury, usually developing within minutes or hours and lasts in hour to few days. It is characterized by five cardinal signs: pain, redness, immobility (loss of function), swelling and heat.
The treatment of inflammation depends on cause and severity, however untreated inflammation can leads to life threatening symptoms. The present invention discloses a treatment based on modulatingthe cause of inflammation, managing symptoms, or both. Currently available drug e.g. non-steroidal anti-inflammatory drugs(NSAIDs), Pain killers and corticosteroids provides only symptomatic relief and does not treat the cause of inflammation. Also being toxic limits their long term usage. Hence there is an urgent unmet need for safer treatment for management of pain and inflammation. Herbal supplements with anti-inflammatory, analgesic and healing properties provides harmless alternative for long term as well as efficacious treatment for management of pain and inflammation. Further careful selection of herbal combination along with other essential metabolic supplements can provide synergistic effect to effectively treat the cause of inflammation to alleviate symptoms. Our present invention discloses such formulation for the treatment of pain and inflammation.
Arthritis is a chronic inflammatory, degenerative disease affecting large number of populations. The progression is mainly related to disability of the joints. There are two types that may arise due to varying symptoms; Rheumatoid arthritis (RA) and Osteoarthritis. RA occurs due the misfunctioning of the body’s own immune system. On the other hand, most common being the osteoarthritis (OA) occurs due to the degeneration the articular cartilage in joints with time. This condition is common in elder people due to the increasing calcification of cartilages/bones and loosening of ligaments surrounding joints resulting in chronic pain, joint stiffness and inflammation. The largest hinge type synovial joint being the knee is most commonly affected with age. The advancement of the disease results not only in the disruption of cartilage homeostasis, it also hampers the non-cartilaginous components of the joint, like the joint capsule, synovium, subchondral bone, ligaments, and periarticular muscles. The subchondral bone starts to thicken, osteophytes which appear due to increased friction in joints starts to appear in OA joints, elevated inflammation in the synovium and uneven enlargement of the joint (Loeser et al., 2012). Under normal conditions the chondrocytes are quiescent and are responsible for the formation of matrix of the cartilage but their activity is altered majorly in OA conditions leading to cartilage degeneration, which is irreversible in nature, specifically at old age. Cartilage heterotrophy is the main factor leading to disruption of the cartilage.

OA is a joint disorder, which affects the entire joint and surrounding tissues especially the articular cartilage. The functioning and the structural integrity of the articular cartilage mainly depends on the matrix organization and its constituents consisting of collagen VI, matrilin, and fibromodulin as part of the pericellular matrix, and the complex network of proteoglycans and collagens II, IX, and XI forming the bulk of the cartilage extracellular matrix (ECM) (Goldring&Goldring, 2016).The smooth tissue avoids friction between the bones during joint movement and maintains the health nature of the joint. The chondrocytes are fully differentiated cells which form the collagen framework with proteoglycans and glycosaminoglycans and also produce lubricin and hyaluronic acid which form a lubricated layer around the cartilage.Due to the natural process of aging, abnormal mechanical stress and encounter of the chondrocytes with the inflammatory cytokines and other harmful molecules, OA articular chondrocytes lose phenotypic instability and hypertrophic differentiation starts to take place(Singh et al., 2019). Stiffness and decreased mobility of the joint makes it difficult for the chondrocytes to gain their nutrition from the synovial fluid. This causes their revival from the quiescent stage and the production of both matrix proteins and matrix-degrading enzymes is at its peak.
Longitudinal research has proven the fact that little is this damage to the articular cartilages associated with the persistent pain in OA patients since the cartilage does not have any nervous tissue (aneural). O’Neill and Felson stated that synovial inflammation (synovitis) and bone marrow lesions (BML) contribute to the predominant symptom of pain during OA(Felson et al., 2016). Non-specific magnetic resonance imaging (MRI) of OA joints shows the presence of irregular lesions below the subchondral bone known as the BML. These abnormal structures are absent in non-painful knees.
Among the various disabilities in the world, osteoarthritis ranks the fifth position in the list. Global prevalence of almost 7% exists affecting about 500 million people worldwide (Hunter et al., 2020). The disease progression mainly includes the damaging articular cartilage, which is the cushiony structure between the two bones. It can occur mainly due to increasing age, accidental damage or other co-morbidities like diabetes. The symptoms comprise of pain and inflammation, which are accompanied by fatigue and loss of mobility. The patients are most commonly prescribed with non-steroidal anti-inflammatory drugs (NSAIDs), which only provide a symptomatic relief. Unfortunately, rare pharmacological modalities have proven to be highly effective due to momentary symptomatic relief and no cure, less patient compliance. The biggest challenge that arises while treating such diseases is the possibility of direct drug delivery that is least due to the absence of circulatory system in the joint. Multiple therapies have been studied in the past, low impact physical activity seems to be supported by all the current medical societies while other interventions have shown conflicting findings.
The formulationPL02 of the present invention includes flavonoid rich plant extracts and collagen peptides. The formulationPL02 is non-toxic and highly cost effective in treating osteoarthritis symptoms. The combination not only ameliorates pain and inflammation but also prevented subchondral bone pathology. The subchondral bone plays a major role in the joint health by supporting the articular cartilage and modulating inflammation. It also provides a preventive treatment, which does not allow the progression of the disease. Collagen is not only an essential component of the extra cellular matrix of the joint but also of other connective tissues.
SUMMARY OF INVENTION

The present invention provides for a synergistic formulation PL02 for the prevention and treatment of pain and inflammation related disorders such as osteoarthritis and joint pain. In particular, the present invention refers to a synergistic formulation comprising of two or more plant extracts and collagen peptides. The formulation is safe, non-toxic, highly potent and cost effective in treating osteoarthritis symptoms. Further, the formulation PL02 is anti-inflammatory, chondroprotective and osteoprotective.
In an aspect, the present invention refers to a formulation PL02 for prevention and treatment of osteoarthritis and related diseases comprising:
a) two or more whole plant extracts
and
b) one or more collagen peptides
c) sodium bicarbonate

In another aspect, the whole plant extracts are obtained by hydro alcoholic extraction. In yet another aspect, the hydro alcoholic extraction of whole plantis obtained from Hippophae rahmnoides of family Scrophulariaceae and Rosa indica of family Rosaceae and is present in an amount 10% to90 % by weight of the formulation.

In a further aspect, the plant extracts are rich in flavonoids.
In a further aspect, the collagen peptide is purified from collagen hydrolysate obtained from marine, ovine or bovine sources and is present in an amount 0% to 90% by weight of the formulation PL02.
In another aspect, the present invention refers to a process of preparing the herbal formulation PL02, comprising the steps of :
(i) mixing 1:1 , 3:7 or 7:3 ratio of the plant extracts wt/wt;
(ii) dissolving in sterile hydro-alcoholic (30:70- 70:30) solvent under shaking;
(iii) centrifugation at 10,000 rpm and collecting the supernatant, drying; and
(iv) adding the collagen peptide and sodium bicarbonate
In a further aspect, the present invention refers to a method for prevention and treatment of osteoarthritis and related diseases comprising administering a therapeutically effective amount of the formulation to a subject.
In yet another aspect, the related diseases are selected from osteoartheritis, joint pain, cervical pain, Ankylosing spondylitis, muscular pain, inflammatory bowel disease, hepatitis, obesity, neurodegenerative diseases, diabetes, neuropathic pain, allergy, asthma etc. which have inflammation and pain as primary symptoms.
In another aspect, the formulation is non-toxic, anti-inflammatory, chondroprotective, osteoprotective and improves gut health.
In a further aspect, the present invention refers to an oral dosage form comprising the formulation of the present invention in the form of tablets, capsules, sachet, beads, granules, aggregates, powders, gels, solids, semi-solids, or suspensions.
DRAWINGS
The foregoing summary, as well as the following detailed description of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of assisting in the explanation of the invention, these are shown in the drawings embodiments which are presently preferred and considered illustrative. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown therein. In the drawings:

Figure 1 depicts UPLC-MS chromatogram showing different molecular weight compounds peak as detected in the PL02 formulation.
Figure 2 Shows the formulation designing
Figure 3 shows timeline flowchart for treatment strategy of animals and analysis.
Figure 4 shows toxicity assessment of PL02 in rat after 28 days of treatment.

Figure 5 illustrates the efficacy of PL02 in ameliorating pain and inflammation via reducing cytokine in MIA induced animal model of osteoarthritis.

Figure 6 illustrates chondroprotective property of PL02.

Figure 7 illustrates histology images of H&E and Toluidine blue staining of joint tissue.

Figure 8 illustrates effect of PL02 on subchondral bone using Micro-CT analysis.
Figure 9 illustrates the anti-inflammatory efficacy of PL02 in Carrageenan induced animal model of inflammation.

DETAILED DESCRIPTION OF INVENTION

In describing the invention, the following terminology will be used in accordance with the definitions set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. As used herein, each of the following terms has the meaning associated with it in this section. Specific and preferred values listed below for individual process parameters, substituents, and ranges are for illustration only; they do not exclude other defined values or other values falling within the preferred defined ranges.

As used herein, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.

The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

When the term “about” is used in describing a value or an endpoint of a range, the disclosure should be understood to include both the specific value and end-point referred to.

As used herein, the terms “comprising” “including,” “having,” “containing,” “involving,” and the like are to be understood to be open-ended, i.e. to mean including but not limited to.

As used herein, the term “whole plant extracts” means hydro- alcoholicextract obtained from flowers, leaves, stem, seeds and root of the plant. The whole plant extracts used in the present invention are rich in flavonoids.

As used herein, the term “collagen peptide” means purified collagen peptide from collagen hydrolysate obtained from marine, ovine or bovine sources such as fish, cow buffalo and pig.

As used herein, the term “herbal formulation” means a formulation containing plant extracts and collagen peptide and wherein the ratio of Hippophae and Rosa extract is 1.1or 3:7 or 7:3.

As used herein, the term “herbal formulation” is a formulation comprising one or more herbs in specified quantities to provide in medicinal benefits. The present invention provides for a formulation comprising extracts of Hippophae rahmnoides and Rosa indica in a defined ratio of 1:1 , collagen peptide and sodium bicarbonate.
The formulation of the present invention includes flavonoid rich plant extractsand collagen peptide. The formulation contains extracts of Hippophae rahmnoides of family Scrophulariaceae and Rosa indica of family Rosaceae and the collagen peptide purified from collagen hydrolysate and sodium bcarbonate from commercial source. The formulation is non-toxic and highly cost effective in treating osteoarthritis symptoms. Further, the formulation is anti-inflammatory, chondroprotective, osteoprotective and improves gut. The combination not only ameliorates pain and inflammation but also improves the gut health. The gut plays a major role in the joint health in maintaining the gut: liver: joint axis. It also provides a preventive treatment, which does not allow the progression of the disease. Collagen is an essential component of the extra cellular matrix of the joint and tissues.
In an embodiment, the present invention refers to the present invention refers to a formulation for prevention and treatment of osteoarthritis and related diseases comprising:
a) two or more whole plant extracts;
and
b) one or more collagen peptides
c) Sodium bicarbonate

In another embodiment, the whole plant extracts are obtained by hydro alcoholic extraction. In yet another embodiment, the hydro alcoholic extraction of whole plant is obtained from Hippophae rahmnoides of family Scrophulariaceae and Rosa indica of family Rosaceae and are present in an amount 1% to 90 % by weight of the formulation.
In a further embodiment, the plant extracts are rich in flavonoids.
In a further embodiment, the collagen peptide purified from collagen hydolysate obtained from marine, ovine or bovine or fish sources and is present in an amount 0% to 90% by weight of the formulation.
In another embodiment, the present invention refers to a process of preparing the herbal formulation, comprising the steps of :
(i) mixing 1:1/7:3/3:7 ratio of the plant wt/wt;
(ii) dissolving in water alcohol mixture (30:70) under shacking;
(iii) Centrifugation at 10,000 rpm and collecting the supernatant, drying; and
(iv) adding the collagen peptide and sodium bicarbonate
In a further embodiment, the present invention refers to a method for prevention and treatment of osteoarthritis and related diseases comprising administering a therapeutically effective amount of the formulation to a subject.
In yet another embodiment, the related diseases are selected from osteoarthritis, joint pain, cervical pain, muscular pain, inflammatory bowel disease, hepatitis, obesity, neurodegenerative diseases, diabetes, neuropathic pain, allergy, asthma etc. which have inflammation and pain as primary symptoms.
In a further embodiment, the formulation is non-toxic, anti-inflammatory, chondroprotective, osteoprotective and improves gut health.
In another embodiment, the present invention refers to an oral dosage form comprising the formulation of the present invention in the form of tablets, dispersible tablet, capsules, sachet beads, granules, aggregates, powders, gels, solids, semi-solids, or suspensions.
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. All publications and other references mentioned herein are incorporated by reference in their entirety. Numeric ranges are inclusive of the numbers defining the range.

The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.

EXAMPLES

EXAMPLE 1: Preparation of Herbal Formulation
1.1 Plant extracts were obtained from hydro alcoholic 70:30 extraction of Hippophae rahmnoides family Scrophulariaceae and Rosa indica family Rosaceae.
The extracts were mixed in 1:1 ratio at given range for the Hippophae and Rosa extract, centrifuged and analysed further using UPLC-MS.
The standerdised plant extracts were then mixed with collagen peptide and sodium bicarbonate to make the formulation PL02, this was further analysed using UPLC-MS.

1.2: Ultra Performance liquid chromatography- Mass spectrometry (UPLC-MS)
UPLC works on the van Deemter principle, which describes the correlation between the flow rate and height of chromatogram. The van Deemter states that, “the flow rate of smaller particles is much faster in compare with large particles as well as unfolding the correlation of flow rate and plate height”. It combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry.
The analysis of formulation was performed using ACQUITY UPLC Q TOF MS/MS system (Waters Corp., Milford, MA, USA). The chromatography was carried out with Acquity BEH C18 column (dimension: 100 mm × 2.1 mm, 1.7 µm; temperature: 25°C). The mobile phase consisted of 0.1% formic acid (a), acetonitrile (b), and methanol (c), and its flow rate was 300 µl/min. Sample injection volume was 5 µl. Gradient elution program was optimized as follows: initial 90:10% B: C- and increased to 80:20% in 2 min, 50%–60% B: C for 1–3 min, 30%–70% B: C for 3–6 min, then 10:90% B: C for 1 min, and finally increased quickly to 90%–10% in 7–10 min. MS analysis was performed in positive ion modes. Centroid mode data were collected over the m/z range 100–1000 Da with a scan time of 1 s. Accurate mass and molecular formula denomination was acquired with Waters MassLynx 4.1 software (Waters MS Technologies). The molecular mass of the detected compounds was checked using the online software ReSpect for phytochemicals (Figure 1).
EXAMPLE 2: Safety and Efficacy of Formulation on MIA induced Osteoarthritis mice model

2.1 Monosodium Iodoacetate (MIA) induced OA model
The MIA injection model is a well-established in literature as the articular injection of the MIA develops the symptoms very much parallel to human OA. Pain becomes the predominant symptom, which occurs due to the initial inflammation process (Yamada et al., 2018). Pain elevates with the progression of disease in resting state as well as while joint movement. Intra-articular injection of MIA causes chondrocyte cell death, leading to cartilage degeneration and subsequent subchondral bone alterations such as appearance of bone osteophytes. Pitcher et al. laid out a step-by-step protocol for the intra-articular injection of MIA. Along with joint damage, MIA injection induces behavioral changes referred as mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that can be easily measured and quantified. These alterations resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain (Pitcher et al., 2016). Guinea pig model of OA by MIA injection into the knee joint cavity have also been reported. Micro-CT images gathered one month after the MIA injection demonstrated that the cartilage surface of the treated knee specimens exhibits severe loss and erosion of the cartilage in the medial tibial plateau and femur areas. On the contrary, no such observation was recorded for spontaneous OA model in which the symptoms appeared only after 91/2 months. The histological changes marked the presence of a partially caved in cartilage surface and loosely distributed chondrocytes in MIA-treated knee. The specimens from the MIA treated knee joints had a slightly reduced stainability with toluidine blue, and an irregular cartilage surface was observed (J. E. Kim et al., 2018). This proves that local injection of MIA a rapid and minimally invasive method for developing OA model to study pathology of the disease. Brederson et al. carried out a comparative analysis between the medial meniscal tear (MMT) surgical model and the MIA chemical model of OA in rats. The hypothesis stated that at the dose administered (3 mg/50 µl), the MIA-OA model triggered a more widespread sensitization in both the peripheral and central nervous systems and may be a useful model for investigating modulation of OA related pathways including those responsible for referred pain (Brederson et al., 2018). Research projects aiming at development of new analgesic drugs utilize MIA model of pain to evaluate their effect. MIA in rats produces chronic osteoarthritis pain, pharmacological tests are performed in the early (up to 1 week after MIA) versus late (between 2 and 4 weeks after MIA) phase of the rat MIA model. Ziaei et al. evaluated the analgesic and anti-inflammatory effects of the mixture of topical plant extracts of Lawsoniainermis and Ricinuscommunis on MIA rat model (Ziaei et al., 2016).
However, all the effects of MIA are dose-dependent. Yamada and co-workers analyzed that high dose of injection resulted in an increase in the oxidative stress markers, a decrease in antioxidant activity and increased articular cartilage damage. Moreover, 1.5 mg dose of MIA in rats was enough to elicit an inflammatory process similar to that seen in human knee OA and contributed to the pathophysiology of OA induced by MIA, especially on the induction of the symptoms characteristic of this disease in the rats: persistent pain and knee edema. Furthermore, MIA injection also produced extensive damage to the cartilage of the joint, as evidenced by irregular surfaces, thinning and necrotic areas (Yamada et al., 2018)
Safety & Efficacy of Formulation on MIA Induced Osteoarthritis Rat Model
Animals
All the protocols of this study were approved by Institutional Animal Ethical Committee of National Institute of Immunology New Delhi, India. The inbred Wistar rats (50-120 g) both sex Male & female and acclimatized in our laboratory conditions for 7 days with free access to normal standard chow diet and tap water. They were maintained under standard conditions of temperature (23±2 °C) and a relative humidity of 50%. Prior to administration of treatment, animals were weighed and marked.
Efficacy studies in MIA induced mouse model of Osteoarthritis
The MIA-induced OA mouse model was used to investigate the protective effect of formulation on joint cartilage degradation. The mice were anesthetized with xylazine (10 mg/kg, intraperitoneal) and ketamine (80 mg/kg, intraperitoneal). After local trichotomy, the leg was flexed keeping the knee at a 90° angle; the needle was positioned through the infrapatellar ligament and received a single intra-articular injection of 0.2 mg/10 µl MIA (in 0.9% sterile saline) into the right knee joint cavity using a 29-gauge syringe (Pitcher et al., 2016). The control group was injected with an equivalent volume of saline and administered with sterile Phosphate buffer saline (PBS) of 1X concentration having a pH of 7.4.
All the animals were evaluated for mechanical hypersensitivity pain behavioral test performed before OA induction and at days after the saline or MIA injection as indicated in Fig 2.
As shown in Figure 2 Von Frey hairs a standard technique for analysing mechanical allodynia or pain in the knee joint, was used to assess the pain sensitivity in knee joint. The force bared by animal paw was displayed on a digital meter. Von Frey filaments (North Coast Medical, Inc. CA, USA) have been used to assess the mechanical sensitivity of the hind paw of the animals with knee joint arthritis. Typically, paw withdrawal threshold (PWT) is measured in response to increasing pressure stimuli applied to the plantar surface by von Frey filaments. Animals were removed from their home cages and placed in a Plexiglass cage with a wire mesh bottom. The animals were allowed to acclimate for 15 min (or until exploratory and grooming behaviour declined to a level compatible with behavioural testing). Von Frey monofilaments were applied at a 90° to the mid-plantar of the left hind paw of the rats (ipsilateral side of MIA injection) with a series of monofilaments. The force bared by animal paw was displayed on a digital meter. We analysed the efficacy of individual plant extracts with collagen, collagen alone, formulation with (PL02) and without collagen and compared with NSAID Indomethacin. As shown in Figure 2 the mice could bear a much more weight after the formulationPL02 was administered orally continuously for 7 days and at the end of the study, the mice could bear weight equal or more to that of saline group. Plants extract alone have shown some efficacy however, maximum efficacy was observed with formulation PL02 indicating synergistic effect of Rosehip and Hippophae and collagen in PL02. Thus we selected formulationPL02 containing Rosehip, Hippophae and collagen peptide for detailed study.
MIA induction-The MIA-induced OA rat model was used to investigate the protective effect of formulation on joint cartilage degradation. To induce OA, rats were anesthetized with xylazine (10 mg/kg, intraperitoneal) and ketamine (80 mg/kg, intraperitoneal). After local trichotomy, the leg was flexed keeping the knee at a 90° angle; the needle was positioned through the infrapatellar ligament and received a single intra-articular injection of 0.5 mg/ 25 µl MIA (in 0.9% sterile saline) into the right knee joint cavity using a 29-gauge syringe (Pitcher et al., 2016). The control group was injected with an equivalent volume of saline and administered with sterile Phosphate buffer saline (PBS) of 1X concentration having a pH of 7.4, as mentioned in Table 1.
All the animals were evaluated for mechanical hypersensitivity, spontaneous pain and other behavioral tests were performed before OA induction and at days after the saline or MIA injection.
Table 1: Rat group and assigned treatment with dose, route & regimen
Group Treatment Dose, Route and Regimen No. of Animals
G1- Control (0.9% saline) PBS (1X) 0.5 ml, oral, 28 days 06
G2- MIA PBS (1X) 0.5 ml, oral, 28 days 06
G4- MIA + PL02 Formulation Formulation (600mg/kg) 0.5 ml, oral, 28 days 06

2.1 Toxicity assessment
The oral toxicity study of the formulation was carried out as mentioned below in Table 2. After dosing, food was withheld for a further 3–4 hours while animals were observed individually during the first 30 min, and then 2, 4, 6 hours post-dosing, and thereafter once daily over 7 days for clinical signs of toxicity such as mortality, respiratory pattern, changes in general behaviour, skin, eyes, fur and somatomotor activity. The parameters in general observation were observed (Naked eye, touch, activeness of animal, movement etc.) before treatment and after treatment; there was no specific scoring method or instrument used for observation. During the treatment of formulation general behaviour assessment, response, body weight, food weight, and water intake was observed. The serum parameter of the rat was used for biochemical analysis. After dissection of the rats, essential organs were collected for the histopathological analysis.
Table 2: Behavioral responses and general appearance of rat treated with formulation in sub-acute toxicity study
Observation Control group MIA group Formulation group
Change In skin No effect No effect No effect
Eye color change No effect No effect No effect
Food intake Normal Normal Normal
General physique Normal Normal Normal
Diarrhea Not present Not present Not present
Coma Not present Not present Not present
Drowsiness Not present Not present Not present
Breathing difficulty Not observed Not observed Not observed
Sedation No effect No effect No effect
Tremor Not present Not present Not present

No rat displayed remarkable changes in clinical observations during study period. On orally treating the MIA induced rat daily for 20 days, no treatment-related mortalities were observed in any of the groups.
2.2 Body weight, food and water consumption
Body weights of all groups were recorded before administration of doses, further body weight was taken weekly entire treatment and finally on the day of sacrifice. The amount of food and water intake was recorded daily. The consumed amount of food and water were measured before they were provided to each group, their remnants were calculated next day to get the differences, which were recorded as daily food (g/rat/day) and water consumption (ml/rat/day) (Figure 4A).
Body weight, intake of food & water were recorded for on the day 1,4,7,10,14 and 21 days of experiment. No significant changes in the body weight or the diet are observed in treated groups and are comparable to the control group. After 21 days of oral administration of PL02, the food intake and water consumption were not affected. It indicates that the PL02 did not induce appetite suppression and deleterious effects on growth of rats. Normal health of rats suggested that no significant changes were observed in their physiological as well as metabolic activity (Figure 4B-C).
2.3 Blood analysis
Blood was collected from the retro-orbital region (1.5 ml) of the animals for measurement of biochemical parameters after 14 days and 21 days.
2.4 Biochemical analysis
The body has two essential and vital organs for proper function are liver and kidney. The function of liver and kidney are different one is used for metabolism of intake and other one is used for excretion of waste product. To assess the toxicity of any new compound it is necessary to know the status of these two vital organs, which can be checked through biochemical estimation without sacrifice the rat.
Dry tubes containing collected blood were centrifuged at 3000 rpm at 25 °C for 15 min to obtain the serum, which was stored at -20 °C until the measurement of biochemical parameters (Tulip auto analyzer).Serum glutamic oxaloacetic transaminase (SGOT), Serum glutamic pyruvic transaminase (SGPT) (Coral clinical system), Alkaline phosphatase (ALP) (Coral clinical system), Urea, Creatinine (Coral clinical system) and BUN.
On administration of the formulation, levels or activities of biochemical parameters for kidney and liver were found to be in normal range on day 14 as well as day 21 and showed no significant changes as compared to the control group (Figure 4Di-v).
2.5 Histological analysis
Organs such as heart, liver, kidneys, and brain were excised and fixed in 10% formaldehyde for histological analysis. Paraffin-embedded organs were cut to 5 mm sections and stained with hematoxylin and eosin. Stained sections were visualized and all measurements performed using a Nikon eclipse 80i microscope equipped with digital sight microscope camera connected to a computer where the images were transferred and analysed with the NIS Elements Imaging software D2.30, SP4 (Build 387)
Histology of essential organs such as liver, heart, kidney,brain,and skin was performed to check if the combination had any adverse effect on the essential organ. No lesions in the tissues were observed (Figures 4E).
6.6 Measurement of knee oedema
The circumference of the MIA injected knee was measured using a Vernier calliper. The readings were noted on the day of experiment before MIA injection and also on following days. Comparison of the differences between the inflamed and right knee joint width was done. The knee joint width was measured by vernier calliper in different days 1, 3, 7, 10, 14, 21 for each group. Treatments were continued for 21 days. On day 1, the difference in the knee joint in both disease control as well as the formulation group is significantly much higher than the saline injected group. This indicated a successful development of the model by an inflamed knee joint. However, in the formulation treated group, the inflammation starts to decrease in the joint from 7 onwards and at day 21, significant fall in the knee joint width is seen which is closer to the saline group. In the disease control, the inflammation in the knee joint still exists at the end of the experiment).
Inflammation in the knee joint has reported to be a major symptom of osteoarthritic joint. Intra-articular injection of MIA leads to the damage of articular cartilage resulting in inflammation in the knee joint. The effects start to show within 24 hours of injection. On the first day of evaluation and behavioral assessment, we observed remarkable increase in the knee diameter as compared to the control group, which was injected with 0.9% saline. This indicated the successful development of the model. The formulation was orally administered to the treatment group and the difference knee diameter was calculated using a vernier caliper. In the formulation treated group, the inflammation starts to decrease in the joint from day 7 onwards and at day 21, significant fall in the knee joint width is seen which is closer to the saline group. In the disease control, the inflammation in the knee joint still exists at the end of the experiment (Figure 5A).
2.7 Plethysmometer
This method was performed as per the previously reported method (Fereidoni et al., 2000). A cylinder filled with mercury was placed on a sensitive digital balance. The values on the digital balance were recorded. According to the gravity of mercury, the expected measures were calculated and compared with the observed value.
Inflammation is also observed in the paw of the injected leg. There were no significant changes observed in the volume of the paw as observed using plethysmometer. This validates the anti-inflammatory property of the formulation (Figure 5B).
2.8 Von Frey test (Pain sensitivity)
Von Frey hairs being the gold standard technique for analysing mechanical allodynia or pain in the knee joint, was used to assess the pain sensitivity in knee joint. The force bared by animal paw was displayed on a digital meter. Von Frey filaments (North Coast Medical, Inc. CA, USA) have been used to assess the mechanical sensitivity of the hind paw of the animals with knee joint arthritis. Typically, paw withdrawal threshold (PWT) is measured in response to increasing pressure stimuli applied to the plantar surface by von Frey filaments. Animals were removed from their home cages and placed in a Plexiglass cage with a wire mesh bottom. The animals were allowed to acclimate for 15 min (or until exploratory and grooming behaviour declined to a level compatible with behavioural testing). Von Frey monofilaments were applied at a 90° to the mid-plantar of the left hind paw of the rats (ipsilateral side of MIA injection) with a series of monofilaments. The force bared by animal paw was displayed on a digital meter. The rats could bear a much more weight after the formulation was administered orally continuously for 7 days and at the end of the study, the rats could bear weight equal to that of saline group. This indicated that the formulation showed therapeutic effects in terms of analgesia (Figures5C).
2.9 Cytokine and chemokine analysis
After 28 days treatment blood was withdrawn from the rats, used for cytokines assay using multiplex analyzer (Figure 5D). Serum levels of proinflammatory and anti-inflammatory cytokines were analyzed using the Bio-Plex ProTMCytokine Standard kit (Bio-Rad, California, USA). The plate was prepared following the instruction manual provided by the manufacturer and read using Bio-Plex 200 Systems (Bio-Rad, California, USA). The data was analyzed on Bio-Plex Manager (acquisition and Analysis) software at low PMT, RP1 instrument settings. The level of TNF-a was significantly (p<0.0001) decreased in MIA + PL02 group comparison to MIA. The level of IL-1ß also decreased but it was not significant after analysis in the PL02 treated group in comparison to the MIA group. Same pattern found in the IL-10 and IL-17 levels, the group treated with PL02 showeda lower level in comparison to the MIA group, but it was not found significant after analysis. However, the level of IFN- ? showed significantly (p<0.0001) lower levels in MIA+ PL02 group in comparison to the MIA group.
To further validate the data real time PCR analysis of pro-inflammatory cytokines IL-1ß and TNF-a was carried out at mRNA level in the knee joint having complete synovial capsule to evaluate the effect of formulation on OA. The relative expression of specific messenger RNA (mRNA) was quantified by real-time polymerase chain reaction (PCR) using SYBR Green I (Roche Diagnostics, Mannheim, Germany). The sense and antisense primers used are given in Table-3.The mRNA expression of pro-inflammatory mediators IL-1ß (Figure 5F) and TNF-a (Figure 5E) decreased significantly in the PL02 treated rats compared with the MIA group (p<0.05) and (p<0.0001), respectively. Chemokines like CCL-2 (Figure 5G) and CXCL-12 (Figure 5H) also show significantly declined levels (p<0.0001 & p<0.001) after administration of formulation for 28 days daily in the PL02 treated group in comparison to MIA group.

2.10 chondroprotective efficacy of PL02
The effect of PL02 on the oxidative stress biomarker, LPO, was determined in serum samples collected from the different treatment groups of animals on 28th day post treatment (Figure 6A). Lipid peroxide, a biomarker for oxidative damage in cells, was measured in the rat serum using ELISA kit (Immunotag™ G-Biosciences, Missouri, USA). Lipid Peroxidation (LPO) Assay was performed according to the instruction manual provided by the manufacturer. The serum level of LPO in the MIA group was significantly increased to 8.302 nmol/mL as compared to control (5.494 nmol/mL). However, the PL02 treatment markedly reduced serum levels of LPO to 6.291 nmol/mL. The relative expression of specific messenger RNA (mRNA) of NOS-2, BCL-2, MMP-13, Aggrecan, TIMP-2, Sox-9 were quantified by real-time polymerase chain reaction (PCR) using SYBR Green I (Roche Diagnostics, Mannheim, Germany). The sense and antisense primers used are given in Table-3. In real time PCR analysis of whole joint tissuethe significantly decreased level (p<0.01) of NOS-2 (Figure 6C) was found in the PL02 group in comparison to the MIA group. The significant high level (p<0.0001) of BCL-2 confirmed the anti-apoptotic property of the PL02 (Figure 6B) in comparison to MIA. This validates the antioxidant and anti-apoptotic property of the PL02 formulation.
Next, we checked the level of protease, mainly Matrix metalloproteinase 13(MMP13) that attacks the cartilage matrix leading to its destruction. The MMP-13 expression increased post MIA injection compared to control. The significant down regulation in the level of MMP-13 was observed in PL02 treated animals compared to MIA (Figure 6D). Same pattern is also has shown in TIMP-2 (Figure 6E), an inhibitor of MMPs family involved in the disease process such as arthritis and metastasis. We have also checked the expression level of collagen-II, the main collagen and, aggrecan, a major proteoglycan in articular cartilage, indispensable for maintaining hydration which is needed for proper functioning (load bearing properties) of articular cartilage. The loss of aggrecan shows the characteristic of OA, we observed significant decrease in aggrecan level post MIA injection, and not significant improvement seen in animals treated with MIA+ Indomethacin. Interestingly, 28 days treatment of PL02 showed (Figure 6G) a significant high-level expression (p<0.0001) of aggrecan in comparison to MIA. Similarly, significant decrease in the expression of Col-II gene in MIA group was observed compared to control. A significantly increased expression Coll-II was observed in the PL02 treatment group compared to the MIA group. However, no significant change was observed in the Indomethacin treated group in comparison to MIA. The expression of COL-II and Aggrecan in the PL02 treated group was comparable to control.
The increased Aggrecan and Coll-II gene expression indicate healthy chondrocytes. Subsequently, to confirm that we determined the mRNA expression level of SOX-9, a master regulator of chondrogenesis. The high level of SOX-9 confirms proliferating but not hypertrophic chondrocytes and is essential for differentiation of precursor cells into chondrocytes. The SOX-9 level decreases significantly post MIA injection compared to control. Treatment with indomethacin did not show any improvement in SOX-9 expression. Whereas, the 28 days treatment with PL02 in rats showed a significant high level of SOX-9 (Figure 6F) in comparison to MIA. The increased SOX-9 expression is corroborated with increased aggrecan and COL-II expression which clearly indicate chondroprotective as well as regenerative properties of PL02.
Table 3: List of Primer with sequence used in Quantitative Real time PCR analysis

2.11Histology of knee joint
The key cells that play a major role in disease progression are the chondrocytes which are present in the articular cartilage. Histology of the knee joint depicts the actual joint health after the experiments. Histological studies were performed to ensure the responses obtained from the pharmacological experiments. On MIA post day 14, rats were sacrificed and MIA or saline injected knee joints (including distal femur and proximal tibia) were fixed in 10 % buffered formaldehyde solution, decalcified using formic acid-sodium citrate method (Munro, 1971). This method is superior to other decalcification techniques, since preserves the histological and staining properties of tissues very well. The formalin-fixed specimens were washed properly by distilled water to remove the residues of formaldehyde from the tissues. Then, the specimens were transferred into a jar containing sufficient volume of formic acid–sodium citrate solution, prepared as follows Solution A: 50 g of sodium citrate was dissolved in 250 ml of distilled water. Solution B: 125 ml of 90 % formic acid were added to 125 ml of distilled water. To make a working solution, equal volumes of the solutions A and B were mixed before use. Due to higher volume of the bone tissue of the specimens, the decalcifying solution was changed every single day until decalcification was completed. The decalcified specimens were washed very well with distilled water in order to remove decalcified residues and then embedded in paraffin. Sections of the tissue specimens were acquired from the paraffin blocks at 5 µm thickness, deparaffinized, and rehydrated in the order of xylene, absolute alcohol, and 50 % alcohol. The rehydrated sections were stained with hematoxylin and eosin (H&E) for observation of morphological changes. Besides, H& E stain, the sections of knee joints stained with toluidine blue for evaluation of the proteoglycan and glycosaminoglycans content. Stained sections were visualized and captured using USB 2.0 Camera Viewer (Leadzoptics Microscope, England, UK).
The histology was evaluated through double-blind observations
In the control group, where saline was administered into the intra-articular region of left knee joint, the articular cartilage surface was smooth, and the chondrocytes are arranged orderly in superficial, mid and deep zone of cartilage. No proliferative changes of chondrocytes are observed and the chondrons do not appear to be distorted. After the injection of MIA in the MIA group, superficial fibrillation of the cartilage surface along with its loss is seen. Chondrosenesence occurred as a result of MIA injection leading to fragmentation of chondrocytes within the chondron in the deep zone whereas distortion of the complete chondron is seen in the superficial and mid zone of the cartilage. Loss of superficial matrix parallel to the surface and formation of fissures is also evident in H&E-stained distal epiphyseal cartilage of the tibia of the left-knee joint in rats. Damage to the bone due to formation of subchondral bone cyst is observed. In the Indomethacin treated group, the cartilage destruction was restricted and only a few deformations of chondrons were observed. On treating the rats with the PL02 formulation daily, for a period of 28 days shows improved macroscopic changes in the articular cartilage as compared to MIA. The surface was smooth and no irregularities in the cartilage surface are seen and the chondrocytes are arranged in parallel zones in well-structured lacunae (fig. 7A). Next, we studied the level of proteoglycan and glycosaminoglycan in the joint tissue section using toluidine blue staining. The toluidine blue staining showed proteoglycan and glycosaminoglycan content were significantly decreased in the MIA group. Whereas MIA+PL02 treatment protected the cartilage from damage and significantly increased level of proteoglycan compared to MIA group (fig. 7A) was observed. Our histological observations were in line with our gene expression profile and substantiate the chondroprotective nature of PL02.
2.12 Subchondral bone analysis using micro-CT
Before imaging, fixed knee joints were further dissected under a dissection microscope to disarticulate them, carefully remove the menisci, and expose the articular cartilage of the tibia. Both femur and tibia were subjected to Micro-CT imaging and analysis using µCT (Quantum GXII, Perkin Elmer, USA). Scans were performed using the Aluminium 0.5+copper 05 filter with the following parameters: time 14 minutes, Voxel Size 36M3, tube voltage 90 kV, and tube current 180 mA. Sub reconstruction of the acquired image was also performed to generate Sub-volume vox files with a size of 18um. Analyze 14.0 was used to create serial 2D coronal and axial pictures from 3D reconstructed CT scans (Perkin Elmer, USA). We estimated BMD(Bone mineral Density), BV/TV, BV, Tb.Th, and Tb.Sp using analyse14 software by manually selecting the correct region of interest i.e. lateral and medial sub chondral bone in the tibia/femur epiphysis.
After histopathological analysis we went ahead to analyze the subchondral bone microarchitecture using Micro-CT. All the rats treated with MIA showed pathological changes in subchondral bone in the injected knee, whereas the contralateral control knee showed no OA-like changes in the tibial subchondral bone microarchitecture.
Figure 8a shows the data comparison of the tibial subchondral trabecular bone microarchitectural parameters of the control knee, the MIA-injected, MIA+PL02 and MIA+ Indo treated knee after completion of 28 days treatment. We observed more significant changes in the medial compartment of Tibia than lateral, therefore considered medial and total changes for interpretation. There was a significant increase in BV(bone volume) and BV/TV in the MIA-injected tibia compared to the control tibia, in both the medial and the total compartments (-15% and -12%, respectively, P < 0.05 for both parameters). However, no such increase in bone volume fraction was observed in animals treated with PL02 and was comparable to control. The trabecular parameter Tb-Th (trabecular thickness) was slightly increased in MIA treated knee, however, the difference was not significant. A significant decrease in Tb.Sp (trabecular spacing) was observed in MIA treated knees compared to control. This decrease in Tb.Sp was prevented by PL02.
The MIA injected knee showed significant increase in tibial subchondral plate (both medial and total compartments) compared to the control knee. The calculated value for subchondral plate thickness was 140±25 &120±9 µm for MIA injected and control respectively. The subchondral plate thickness 115±15 µm was observed in the PL02 treated knee which is similar to control. Moreover, we have also observed increased porosity of the tibial subchondral plate in the MIA-injected knee compared to the control and PL02 treated knee. The %d MIA-Control was 48 and MIA+PL02-Control was 20, p<0.001.
Fig 8b shows the lateral and coronal view of the tibia. As can be seen in the Micro-CT image, increased sclerosis was observed in MIA injected knees compared to control. The sclerosis reduced upon indomethacin treatment. Interestingly no sclerosis was observed in PL02 treated animals’ knees. Thus, the overall analysis of tibial subchondral bone clearly indicated that MIA injection induced subchondral pathology reported in humans and this was prevented by PL02 treatment.
Example 3
3. In –Vivo Anti-Inflammatory Activity
3.1 Carrageenan Model
Carrageenan, sulphated mucopolysaccharide extracted from red edible seaweeds, is intra-articularly injected into the joint to develop inflammatory OA model. Inflammation being the key pathway behind OA pathology, Carrageenan-induced arthritis in the rat is also a practical model of arthritis. Carrageenan induces arthritis quickly and is simple to prepare, time-saving and administer. A study demonstrated the differences in the control and arthritic groups in terms of articular cartilage thickness of the femur, epiphyseal plate thickness of the tibia and femur, subchondral bone plate thickness of the tibia and synovial lining cell layer thickness. Decreased articular cartilage thickness of the femur in carrageenan-induced OA model may be related to cartilage matrix depletion due to inflammatory arthritis, and increased growth plate thickness may be related to inflammatory arthritis-induced hyperaemia and hypermetabolism of the growth plate. Both of these features are well known in human disease, and have been observed in other carrageenan OA model (Hansra et al., 2000). Intra-articular injection of 2% carrageenan into rat knee joints produces synovitis and pain behaviour measured as hindlimb weight bearing asymmetry (Fehrenbacher et al., 2012). Carrageenan-injection in OA knees also increased cartilage damage and osteophyte maturity (Ashraf et al., 2018). There lies much evidence in the literature about the sudden inflammatory effects of carrageenan just after injection. Oedema is the result of an imbalance in water filtration between the blood vessels and interstitial spaces. Carrageenan injection induced paw oedema which was peaked at 6?h and gradually decreased nearly to the initial baseline value after 72?h (K. H. Kim et al., 2020) (Figure 9).
Experimental Animals
Female C57BL6J mice were used for the present study. The mice were housed in con-trolled-ventilation cabinets at room temperature (22±2°C), controlled humidity at 60-80%, and light-dark cycles (12 hours). The animals were conditioned in acrylic boxes and they received food and water ad libitum. The behavioural assessments were performed during the day light phase. All the procedures used in the present study were approved by the institutional animal care and ethics committee of National Institute of Immunology.
The anti-inflammatory activity of PL02 formulation was evaluated by the carrageenan injection method. 10µL of 1% carrageenan dissolved in 0.9% saline was injected into the left hind paw of the mice.
The mice (Black 6) were divided into three groups:
(a) Control PBS group (n = 5) where the animals received PBS
(b) Carrageenan (Cgn) group where the animals received Indomethacin (20mg/kg of body weight);
(c) Carrageenan + Treatment group where the animals received PL02 formulation.
0.5ml of treatment composed by natural fish collagen and two other plant extracts was administered orally by gavage, once before carrageenan administration.
3. 2 Biochemical analysis
The body has two essential and vital organs for proper function are liver and kidney. The function of liver and kidney are different one is used for metabolism of intake and other one is used for excretion of waste product. To assess the toxicity of any new compound it is necessary to know the status of these two vital organs, which can be checked through biochemical estimation without sacrifice the rat. For liver function assessment mainly used the SGOT, SGPT and ALP however for kidney function assessments mainly used urea and creatinine.
Dry tubes containing collected blood were centrifuged at 3000 rpm at 25 °C for 15 min to obtain the serum, which was stored at -20 °C until the measurement of biochemical parameters (Tulip auto analyzer).Serum glutamic oxaloacetic transaminase (SGOT), Serum glutamic pyruvic transaminase (SGPT) (Coral clinical system), Alkaline phosphatase (ALP) (Coral clinical system), Urea, Creatinine (Coral clinical system) and BUN.
Table 4: Biochemical estimation from blood serum of Rat 48 hour different treatment study
Parameters Normal range Control Indomethacin Formulation PL02
SGOT (U/L) 54-298 78.17± 3.94 89.02 ±3.575 74.585± 0.001
SGPT (U/L) 17-77 59.015 ±1.055 49.445± 8.06 55.95± 7.905
ALP (U/L) 64-128 83.375± 2.285 83.545 ±8.9 90.88 ±0.225
Creatinine (mg/dL) 0.2-0.9 0.305± 0.005 0.245± 0.0045 0.25 ±0.01
Urea (U/L) 35-96 53.06 ± 1.21 55.975 ± 1.79 54.49 ± 1.185
BUN (mg/dL) 8-33 29.44 ±0.56 25.44±0.83 26.14±0.55

On administration of the formulation PL02, levels or activities of biochemical parameters for kidney and liver were found to be in normal range on day 14 as well as day 21 and showed no significant changes as compared to the control group.
3. 3 Inflammation in the injected paw was measured using plethysmometer. This method was performed as per the previously reported method (Fereidoni et al., 2000). A cylinder filled with mercury was placed on a sensitive digital balance. The values on the digital balance were recorded. According to the gravity of mercury, the expected measures were calculated and compared with the observed value. Displacement in mercury gives the reading for inflammation in the paw volume. Measurements of the inflamed paw were done at 0,1,2,4,6,24,30,48 hours after injection ?-carrageenan. The paw volume significantly reduced after 2 hours in formulation PL02 group as compared to PBS control group (Figure 9A).
3.4 Rotarod was performed on the mice of all groups to check for their locomotive activity. The animals were placed randomly on a rotating cylinder with increasing speed, forcing them to walk continuously to avoid falling. The performance index provided motor learning and use of the affected limb. They were kept on the rotating bar for 5 min to become accustomed to the appliance. Five minutes after the adaptation period, they were again placed on Rotarod and the rotational speed was increased from 5 to 35 rpm in range of 5 min. The latency to fall was measured automatically by a mechanical sensor at the base of the device. The time mice spend on the moving rod was measured at 0,1,2,4,6,24,30,48 hours after injection ?-carrageenan compared to saline control group. The latency values were increased after 6 hours in formulation PL02 treated group. A uniform rise in the time spent by mice on the rotating rod increased gradually. The rise is higher in formulation PL02 as compared to Indomethacin,anapproved NSAID (Figure 9B).
, Claims:We Claim:

1. A formulation for prevention and treatment of pain and inflammation and related diseases comprising:
a) two or more whole plant extracts;
b) one or more collagen peptides and
c) sodium bicarbonate.

2. The formulation as claimed in claim 1, wherein the whole plant extracts are obtained by hydro alcoholic extraction.

3. The formulation as claimed in claim 2, wherein the hydro alcoholic extraction of whole plant are obtained from Hippophaerahmnoides of family Scrophulariaceae and Rosa indica of family Rosaceae.

4. The formulation as claimed in claim 1, wherein the plant extracts are rich in flavonoids.

5. The formulation as claimed in claim 1, wherein the plant extracts are present in an amount 10% to 90 % by weight of the formulation.

6. The formulation as claimed in claim 1, wherein the collagen peptide is purified from collagen hydolysate selected from marine, ovine or bovine sources.

7. The formulation as claimed in claim 1, wherein the collagen peptide is present in an amount10% to 90% by weight of the formulation.

8. The formulation as claimed in claim 1, wherein the sodium bicarbonate is present in an amount 10% to 90 % by weight of the formulation.

9. A process of preparing the formulation as claimed in claim 1, comprising the steps of:
(i) mixing 1:1 – 3:7 /7:3 ratio of the plant extracts wt/wt;
(ii) dissolving in sterile water under shaking;
(iii) centrifugation at 10,000 rpm and collecting the supernatant; drying and
(iv) adding the collagen peptide and sodium Bicarbonate.

10. A method for prevention and treatment of osteoarthritis and related diseases comprising administering a therapeutically effective amount of the formulation orally as claimed in claim 1 to a subject.

11. The method as claimed in claim 8, wherein related diseases are selected from osteoarthritis, joint pain, cervical pain, muscular pain, inflammatory bowel disease, hepatitis, obesity, neurodegenerative diseases, diabetes, neuropathic pain, allergy, asthama etc. which have inflammation and pain as primary symptoms.

12. The formulation as claimed in Claim 1, is non-toxic, anti-inflammatory, chondroprotective, osteoprotective and improves gut health.

13. An oral dosage form comprising the formulation as claimed in claim 1, is in the form of tablets, capsules, beads, granules, aggregates, powders, gels, solids, semi-solids, or suspensions.

Dated this 22nd day of August, 2022