Topical Phytotherapeutic Treatment: Management of Normalization of Elevated Levels of Biochemical Parameter During Osteoarthritic Disorders: A prospective Study

Background: C-reactive protein (CRP), creatine kinase-muscle (CK-MM) and aldolase A (AldoA) levels are proven to be realistic biochemical markers of osteoarthritic disorders (OADs). The aim of this study is to normalize the elevated levels of CRP, CK-MM and AldoA during OADs of knee-joints and degenerative changes in lumbarspine by topical application of phytoconstituents with specialized techniques. Methods: Baseline data were collected and evaluated from 261 patients (66.67% females) with OADs confirmed with radiological images. Serum CRP, CK-MM, and AldoA levels were measured at the baseline and at the end of six weeks of treatment using appropriate kits. All patients underwent a standardized physical, radiographic examinations and evaluation of risk factors (inflammation, muscle degeneration, bone erosion, and skeletal muscle diseases) by blood examination and completed a questionnaire. All the patients were treated with topical application of phytochemicals extracted from seven Indian medicinal plants mixed with virgin sesame oil and beehive-wax for a period of six-week with specialized techniques. Results: The parameters of the risk factors for OADs at the end of six-week of treatment were highly significant (p<0.0001). The high and low ranges of all biomarkers, their percentage of improvements and Pearson’s correlation coefficient were all highly significant (p<0.0001). The improvements of pain, stiffness and daily functional activities at the end of treatment under KOOS, VAS, WOMAC index and KPS were highly significant (p<0.0001) and also reduction of weight as evidenced by BMI (p<0.0001) and radiological images as assessed by K-L grading score. Conclusions: It is firmly confirmed from the results that the elevated levels of CRP, CK-MM, and AldoA, represent the risks of inflammation, muscle degeneration, and skeletal muscle damage along with pain and all daily functional activities, body weight and radiological images as confirmed by above-mentioned international approved scales during OADs can be successful normalized by topical application of phytoconstituents within six-week with specialized treatment.

In general, the elevated levels of serum C-reactive protein (CRP) in patients is found during inflammation, creatine kinasemuscle (CK-MM) due to muscular dystrophy, connective tissue damage, etc. and Aldolase A (AldoA) due to skeletal muscle or liver damage [18][19][20][21][22][23][24][25][26][27]. In this context, no synthetic medicines are found to normalize these elevated levels of biochemical parameters but topical phytotherapy are well-known to normalize these parameters supported by anatomical and radiological features [11,[28][29][30]. Although, several phytomedicines are well-established for the curing of diseases and biochemical mechanisms of action by plant derived natural constituents such as phytochemicals have already been reported by several researchers [11,.
Moreover, there are well-known guidelines for the standard clinical outcomes for pain and related parameters such as visual analogue scale (VAS), Western Ontario and McMaster Universities Osteoarthritis (WOMAC) Index, and Knee injury and Osteoarthritis Outcome Score (KOOS) used for assessing the effect of novel treatments on OADs by several researchers [65][66][67]. In case of gradation of narrowing of joint space, osteophytes lipping, sclerosis and deformity of bone contour under radiological investigation, the Kellgren-Lawrence grading scale is a common method to classify the severity of knee OA with the help of five grades (0-4) [68].

Study design
After the analysis of exclusion criteria including discontinued (dropped out) patients, the balance of 261 (174 females and 87 males) patients with symptoms of visual inflammation, pain and restricted movements, symptomatic knee arthrosis, degenerative changes in lumber region according to the radiological analysis were finally involved in the study summarized in Figure 1. Separate evaluations were performed for subjects regarding various complaints and supplements taken to diminish pain or improve fitness. The present studied protocol has been designed to control pain, to maintain joint flexibility, to optimize joint and limb functions and to improve quality of life without any kinds of drug therapy such as acetaminophen, NSAIDs, including COX-2 inhibitors, corticosteroids and hyaluronic injections, glucosamine sulphate, chondroitin, calcium and other alternative treatments such as homeopathic, ayurvedic and hence patients were advised to stop using any kind of above-mentioned drug therapy one day prior to inclusion in the study. Each patient completed a questionnaire, providing details regarding demographics, medical history, nutritional status, ethnic barriers and work status at the baseline and summarized in Table 2.

Evaluation of biochemical parameters in blood
A 5 ml blood sample was collected in a plain vial from each patient with OAD before and after the treatment. Blood samples were then centrifuged at 1000 g for 10 min at 4 °C to obtain serum. Finally, the serum was used to analyse CRP, CK-MM, and AldoA levels for each

A)
144 patients (94 females and 50 males) were excluded for the following reasons with: 1. Cuts, wounds or any type of chronic skin and infectious disease (12 females and 6 males).

2.
Parallel multiple drug dependence for concomitant diseases or risk conditions requiring drug treatment including psychiatric diseases etc. (15 females and 7 males).
A history of severe neurological diseases (13 females and 7 males) 7.
A history of chronic liver, kidney and heart diseases ( 12 females and 6 males) and 8.
Patients who did not agree to a physical evaluation may be due to drugs/alcohol addiction, excessive over weight, pregnancy and such other reasons (13 females and 7 males). B) 38 patients (21 females and 17 males) were also additionally excluded for having another pathological condition that could explain the existing symptoms such as: 1. Rheumatic diseases, 2.
Joint symptoms caused by malignant tumors, 7.

Discontinued (dropped out) during treatment
25 patients (14 females and 11 males) were discontinued their treatments in the clinic for the following reasons : general practitioner or specialist were allowed to use NSAIDs or other rescue medications, problems for transportation; shortage of helping hands at old age; unable to apply medicines at home, twice/thrice a day and transferring the jobs in other city as under: a) 7 patients after a week (4 females and 3 males) b) 7 patients after second week (5 females and 2 males) c) 6  CRP levels (mg/L) were quantitatively assessed using a CRP LEIT kit and the latex-enhanced turbidimetric immune assay (Agappe Diagnostics Limited, India) at a wavelength of 578 nm. The kit was developed based on the methods reported by Tillett WS et al., Ziegenhagen G et al. and Rifai N et al. [69][70][71]. CK-MM (U/L) levels were quantitatively assessed using a Creatine Kinase-MM kit (CK-MM/CPK-MM/CK-3) and an immunoassay (Aalto Scientific, Limited, USA). The kit was developed based on the methods reported by Cabaniss CD [72]. AldoA levels (U/L) were quantified using an ALDOLASE (ALS) RX MONZA AD 189 kit (Randox Laboratories Ltd, Antrim, UK) based on a photometric assay at a wavelength of 340 nm. The kit was developed according to the method reported by Feissli S [73]. The subjects suffering from OADs with inflammation, muscle degeneration and skeletal muscle damage were studied to identify a specific biochemical parameter, such as CRP, CK-MM, and AldoA levels, in the affected population. All blood tests were conducted under the supervision of the Chief biochemist and the Chief pathologist in the Galaxy Medical Centre, an ISO 9001: 2015 certified lab with Registration No. L/004-(05)-15/0129 under the W.B Clinical Establishment Act 1950.
The mean, standard error of the mean (SEM) and their mean differences (MDs), 95% confidence intervals (CIs), and p-values of the three biomarkers such as CRP, CK-MM and Aldo A were evaluated at the baseline and at the end of six weeks of treatment.

Evaluations of the high and low ranges of biochemical parameters and their percentage of improvements
The reference ranges of the three biomarkers are as follows: CRP<6 mg/L, CK-MM<168 U/L and AldoA<7.6 U/L. The 261 participants were divided into six categories according to the high and low ranges of the three biomarkers: CRP<6 mg/L and CRP>6 mg/L; CK-MM<168 U/L and CK-MM>168 U/L; and Aldo A<7.6 U/L and Aldo A>7.6 U/L. The number of patients and their percentages in the high and low ranges of the three biomarkers such as CRP<6 mg/L, CRP>6mg/L, CK-MM<168.0U/L, CK-MM>168.0 U/L, Aldo A<7.6 U/L and Aldo A>7.6 U/L were studied. The mean, standard error of the mean (SEM) and their mean differences (MDs), 95% confidence intervals (CIs), and p-values of the three biomarkers in the high and low ranges were evaluated at the baseline and at the end of six weeks of treatment.

Evaluation of Pearson's correlation coefficients among two biomarkers
The Pearson's correlation coefficients among two biochemical markers such as CRP at the baseline (CRP b ) and CK-MM after the treatment (CK-MM t ), CRP at the baseline (CRP b ) and Aldo A after the treatment (AldoA t ), CK-MM at the baseline (CK-MM b ) and AldoA after the treatment (AldoA t ), CRP at the baseline (CRP b ) and CRP after the treatment (CRP t ), CK-MM at the baseline (CK-MM b ) and CK-MM after the treatment (CK-MM t ), and AldoA at the baseline (AldoA b ) and AldoA after the treatment (AldoA t ) were studied.

Evaluation of KOOS knee survey
The five separately scored subscales such as pain, other symptoms (swelling, restricted range of motion and mechanical symptoms), function in daily living (disability on the level of daily activities), function in sport and recreation (disability on a level physically more demanding than activities of daily living) and knee-related quality of life (quality of life, mental and social aspects such as awareness and lifestyle changes) under Knee-injury Osteoarthritis Outcomes Scale (KOOS) developed by Ewa Roos and co-authors in the 1990s to assess the patient's opinion about their knee and associated problems as an instrument were evaluated at the baseline and at the end of six weeks of treatment for all the patents.

Evaluation of pain under Visual analogue scale (VAS)
Observation of patient's perceived symptoms of pain intensity was point out on the line of 100 mm. Their pain intensity marked as no pain (0-4), mild pain , moderate pain  and severe pain (75-100) separately on right leg, left leg and lower back pain under Visual analogue scales (VAS) were evaluated before and after the treatment and their percentage of improvement were also evaluated.

Evaluation of WOMAC osteoarthritis Index
The assessment of pain, stiffness and physical functional disabilities of each patient were evaluated for all females and males separately using The Western Ontario McMaster Universities Osteoarthritis Index (WOMAC Index) developed by Bellamy N et al. [66] at the baseline and end of 6-week and their percentage of improvement were studied.

Evaluation of the Karnofsky performance status (KPS) scale
Karnofsky performance status (KPS) score is used to determine a patient's prognosis to carry out daily activities. A higher score indicates the patient is better able to carry out daily activities and its range from 0 to 100. The KPS is evaluated for each patient at the baseline and at the end of treatment. The percentages of improvements were evaluated at the end of the treatment for all the patents separately. Their mean, SD and p-values for overall and separately by gender were studied.

Evaluation of Body mass index (BMI)
Body weight in kilogram was measured without shoes or heavy clothing using an electronic scale. Height in meter was measured without shoes using a wall-mounted stadiometer. Body mass index (BMI, kg/m 2 ) was calculated for all the patients based on measured weights and heights at the baseline and at the end of 6 weeks of treatment. The percentages of improvements were evaluated at the end of treatment for all the patents separately.

Evaluation of radiological images with the Kellygren and Lawrance system
The Kellgren and Lawrance system is a common method of classifying the severity of knee osteoarthritis (OA) developed by Kellgren JH et al [68]. The KL classification was originally described using anterior-posterior (AP) knee radiographs based on the five grades such as Grade 0, which demonstrates no radiographic features of OA present; Grade 1, which demonstrates doubtful joint space narrowing (JSN) with possible osteophyte lipping; Grate 2, which demonstrates possible JSN with definite osteophyte formation; Grade 3, which demonstrates definite JSN, moderate osteophyte formation, some sclerosis and possible deformity of bony ends and Grade 4, which demonstrates large osteophyte formation, severe JSN with marked sclerosis and definite deformity of bony ends. Radiological images for both knee joints and lumbo-sacral spine of 261 combined patients were collected at baseline and at the end of six weeks of treatment evaluations The KL classification of AP knee radiographs of all patients were evaluated at the baseline and at the end of 6-week treatment.
The AP knee and lumbar vertebrae X-ray images of two such patients, before and after the treatment are depicted in Figure 2A-2B.

Evaluation of Indian medicinal plants, their phytoconstituants from aqueous extracts and their established mechanisms of action used in the treatment protocol
The treatment involves topical application of phytoconstituents from the extracts of seven Indian medicinal plants namely Cissus quadrangularis (whole plant), Calotropis gigantea (root and leaves), Zingiber officinalis (rhizome), Rosemarinus officinalis (leaves and flowers), Boswellia serratia (resin), Curcuma longa (rhizome) and Withania somnifera (root) mixed with virgin sesame oil (extracted from seeds at room temperature) and beehives wax to make viscous phyto-based oil without using any preservatives or chemicals in order to preserve the phytochemical properties of plants intact . The virgin sesame oil is acted as bio-preservative and beehive-wax helps to reduce joint pain, to relieve stiffness, to stimulate circulation and to moisturize skin [57][58][59][60][61][62][63][64]. Several researchers had already reported the medical effects (specially on pain, inflammation and stiffness of muscles) on human body of the phytochemicals contained in Cissus quadrangularies, Rosemarinus officinals, Calotropis gigantea, Zingiber officinalis, Boswellia serrata, Curcuma longa, Withania somnifera and Sesamum indicum and their mechanism of actions is shown in Table  3  .

Objectives and phytotherapeutic treatment procedure as a whole
The main objectives of the treatment are: to reduce pain, inflammation and stiffness of muscles without dependence upon any types of pain killers or corticosteroid injection or arthrocentesis; to increase the muscular strength without using supporting belt on the waist or knee joints and the gaps between the bones/vertebrae; to rectify calcifications/degeneration of bones; and to normalize the varus/valgus deformities and elevated levels of biochemical parameters such as CRP,CK-MM and Aldo A occurred during OADs [8,9,11,28,29]. The treatment protocol is based on well-defined certain principles and theories and also based on the applications of wellknown chemical, mechanical, thermal and electrical stimuli which improve the fundamental properties of all muscles such as excitability, conductivity, contractibility, elasticity and viscosity [8,9,11,28,29]. Each 30 ml of said viscous phyto-based oil prepared from the extracts of seven Indian medicinal plants mixed with virgin sesame oil and beehive-wax is to be applied with the tip of three fingers in particular technique over the skin three times a day with minimum interval of two hours for six weeks ; lying in six different postural positions such as supine, prone, right and left contra-lateral and right and left cross (supper) contra-lateral in different programmed sequences in order to nourished the effected group of badly damaged muscles and nerves in the legs and lumber regions during OAD (Table 4) [8,9,11,28,29].
To achieve the ultimate objectives of the treatment, the period Table 3: Medicinal plant species usage for therapy through biochemical mechanism in OAD patients.
Liposaccharide-S induced synthesis of pro-inflammatory cytokines (TNF-α, IL-1β and IL-12) in peripheral and synovial fluid mononuclear cells from rheumatoid arthritis (in vitro), NF kBsubjects in vitro, (-)TNF-α, IL-1β, IL-12, collagenase Anti-bacterial, Anti-oxidant, anti-Inflammation and analgesic, scores for pain, stiffness and disability [25,26] Sesamum indicum (Seed) lower plasmatic levels of thiobarbituric acid reactive substances and reduced gamma-glutamyl-transferase activity in the jointsand spleen, lowering the levels of IL-1β, IL-6), and of high-sensitivity C-reactive protein (hs-CRP) and IL-6impeding the activity of nuclear factor-κB (NF-κB), decreasing serum levels of malondialdehydeinflammatory mediators (TNF-α) of treatment has been fixed for six weeks, three sessions a day. One session in the clinic and two sessions a day with a minimum interval of two hours in the house or thirteen weeks at home (three sessions a day with a minimum interval of two hours). In the clinic, after the application of phytochemicals contained paste with the help of wooden device (WD) and tip of three fingers, number of medicated fomentation devices (MFDs) with control temperature at 106 °F were rapped from the origins to the insertions over the muscles affecting the movement of knee joints and lower spine in different postural positions such as supine, prone, right and left contra-lateral and right and left cross contra-lateral in the first phase of treatment [8,9,11,28,29]. In the second phase of treatment, a computerized muscle stimulator operated with battery of 9-volt DC was applied over the various connective tissues through the skin for a maximum period of 5 to 10 minutes. These helped to disperse the coagulated blood or effusions might be present in the affected areas of OAD patients. In the house treatment of thirteen weeks, only phytochemicals contained paste with the help of wooden device and tip of three fingers is to be applied, three sessions a day with programmed sequences of different postural positions supplied in advanced according to the conditions of OAD of the patients [8,9,11,28,29]. Each patient has been requested to visit the clinic for review in every week. Sometimes depending upon the severity of the pain as well as condition of the deformities, the sequence of the programmed postural positions was altered restricted to two positions in a session. Based on observations for optimum results had tried on more than thousand patients, the maximum period of treatment had been fixed for six weeks (one session in the clinic or two sessions in the house with two-hourly interval) or thirteen weeks at home (three sessions a day with a minimum interval of two hours) [9,11,28,29].

External study reviewers
All results and data before and after the treatment were evaluated by an external reviewing panel consisting of two biochemists, a biostatistician, a radiologist, and a general medical practitioner, none of whom were in contract with the patients.

Data collection and Statistical analysis
Data were summarized using descriptive statistics for continuous variables (e.g. mean, standard error of the mean, N, minimum, maximum), frequency tables, or proportions for discrete variables, and 95% confidence intervals. Statistical analyses were done by using software (Graph Pad Prism, Version 5.0) with repeated measures for student-t test to determine significant values at p<0.05 level along with r (Pearson's correlation coefficient) values to determine strong and weak correlation among two variables for measuring different improvement parameters of OAD patients for combinedsex patients, females and males separately. The comparison was done between baseline and after six weeks of phytotherapeutic treatment.

Patients enrollment and baseline characteristics
A total of 468 patients (68.74% females) those who were suffering with OAD confirmed by X-ray or MRI were included in this study. Figure 1 shows the flow chart of the 261 combined-sex patients (178 females and 78 males) who were finally participated with specific areas of pain in this study after exclusion of 207 patients (Table 1). Table 2 displays the baseline demographic characteristics of all patients. The patients were not being treated by oral medications; injections and any type of alternative interventions such as homeopathic, ayurvedic, natural and herbal treatments for diminishing pain or inflammation, for muscle relaxation or for improvement of skeletal muscles during two weeks and had not undergone arthrocentesis within four months prior to the blood tests such as CRP, CK-MM and AldoA evaluated at the baseline and at the end of the topical phytotherapy treatment of 6-week.

Biochemical parameters
In this study, the biochemical parameters such as CRP, CK-MM and Aldo A were measured at the baseline and after 6-week of phytotherapy treatment from the serum of all participants. Each test for every patient has been rechecked by the S-240 Mindray fully automated biochemistry analyzer before reporting the final test result at the baseline and at the end of six weeks of treatment. Table 5 shows the results which indicated decreasing levels of CRP, CK-MM and Aldo A when compared with the levels at the baseline with the levels at the end of six weeks of treatment considering 174 females and 87 males separately or together and that these differences were highly significant (P<0.0001) for all the parameters either together or separately by gender. Percentage of improvements after the end of six weeks of treatment for all the biomarkers such as CRP, CK-MM and AldoA were highly significant (p<0.0001) in case of combined-sex, female-only and male-only, which were shown in Figure 2.

The high and low ranges of biochemical parameters and their percentage of improvements
The reference ranges of the three biomarkers are: CRP<6 mg/L, CK-MM<168 U/L and AldoA<7.6 U/L. All the participants were divided into six categories according to the high and low ranges of the three biomarkers: CRP<6 mg/L and CRP>6 mg/L; CK-MM<168 U/L and CK-MM>168 U/L; and AldoA<7.6U/L and AldoA>7.6 U/L. The number of patients and their percentage of six categories for combined-sex, female-only and male-only are shown in Table 6. Table 7 shows that the three biomarkers of all the six categories of patients based on high and low ranges were highly statistically significant (p<0.0001) both overall and separately by gender, when compared the baseline with the results at the end of six weeks of treatment.      Figure 4C shows the graphical representation of high and low ranges of CRP, CK-MM and Aldo A levels of 87 male-only subjects. The percentage of improvements in their high and low ranges of six categories including CRP<6.0 mg/L, CRP>6.0 mg/L, CK-MM<168.0 U/L, CK-MM>168.0 U/L, AldoA<7.6 U/L and AldoA>7.6 U/L after the end of treatment of six weeks were highly significant levels (p<0.0001).

Pearson's correlation coefficient
All the Pearson's correlation coefficients among two biochemical markers were weak but highly significant (p<0.05): CRP at the baseline (CRP b ) and CK-MM after the treatment (CK-MM t ) was r=-0.123 (p=0.018) and shown in Figure 5A; CRP at the baseline (CRP b ) and Aldo A after the treatment (Aldo A t ) was r=-0.051 (p=0.034) and shown in Figure 5B; CK-MM at the baseline (CK-MM b ) and Aldo A after the treatment (AldoA t ) was r=0.215 (p=0.000) and shown in Figure 5C; CRP at the baseline (CRP b ) and CRP after the treatment           (CRP t ) was r=0.243 (p=0.000) and shown in Figure 5D; CK-MM at the baseline (CK-MM b ) and CK-MM after the treatment (CK-MM t ) was r=0.289 (p=0.000) and shown in Figure 5E and Aldo A at the baseline (AldoA b ) and AldoA after the treatment (AldoA t ) was r=0.234 (p=0.000) and shown in Figure 5F.

KOOS knee survey
The improvements of the five separately scored subscales under KOOS knee survey after the end of six weeks of treatment were highly significant (p<0.0001) and shown in Figure 6A.

Percentage of improvement of pain, stiffness and daily functional activities under other three International approved scales and reduction of body weight
The percentage of improvements of pain, stiffness and daily functional performances under VAS, WOMAC Index and KPS were highly statistically significant (p<0.0001) both over all and separately by gender and shown in Figures 6B-6D. Figure 7 shows the percentage of reduction of body weights of combined-sex, female-only and male-only patients after the end of six-week treatment protocol as assessed by BMI were highly statistically significant (p<0.0001).

Improvement of OAD as per radiological images
All the anterior-posterior (AP) X-ray reports of 261 patients with OAD at the baseline exhibited degenerative changes, particularly in the medial tibio-femoral compartment, with marked joint space narrowing and bilateral varus/valgus deformities. Some cases exhibited near-complete medial compartment joint space obliteration and also degenerative changes with osteophytes in the lumbar vertebrae. The AP knee X-rays and lumbar vertebrae of patients after six weeks of treatment indicated no such degenerative changes. X-rays images of two such patients suffering with pain in the knee joint and lumbar region before and after the treatment are depicted in Figures 7A-7B. Table 4 shows the improvement of the radiological images before and after the treatment under the K-L grading scale.

Discussion
In the present study, the importance of the risk factors for OAD i.e. elevated levels of certain biochemical parameters such as CRP, CK-MM and Aldo A in the serum of OAD patients has been taken care for diminishing their levels with the help of topical phytotherapy treatment. The reference value for each parameter in adults has been established as <6 mg/L for CRP, <168 U/L for CK-MM and <7.6 U/L for AldoA. CRP levels increase due to inflammation. CK-MM levels are elevated in response to muscular dystrophy, connective tissue damage, etc. and AldoA levels increase due to skeletal muscle damages and bone erosion, suggesting that these markers may be risk factors for OADs [18,19,21,22,26,[65][66][67][68]. However, previous studies were conducted on the individual elevated level of biochemical marker either CRP or CK-MM or AldoA in separate subjects by several researchers [20,24,27,72] but so far no study has attempted to correlate the risk factors, such as muscular damage, skeletal muscle damage and inflammation, with other diseases, such as OADs.
In recent era, plant derived phytochemicals are called phytomedicines. The biochemical mechanism of actions of these phytomedicines for osteoarthritis (OA) prevention is reported by several researchers 72]. Moreover, Dragos D et al. [54] documented that phytochemicals extracted from several plant species could be prevented the joint disorders with mechanism of their actions. The joint disorders are mainly related to osteoarthritis (OA), rheumatoid arthritis (RA) and finally leading to inflammation, muscular damage, skeletal muscle damage, bone degeneration etc. All of these above-mentioned diseases cause alteration of biochemical mechanisms of action such as enzymatic changes in blood, synovial fluid and in different cell types by different clinical, in vitro and in vivo studies [54].
In the present study, it was observed that three biochemical markers in serum resulted a significantly decreasing level when phytomedicines applied through aqueous phytoextracts of Cissus quadrangularis (whole plant), Calotropis gigantean (root and leaves), Zingiber officinalis (rhizome), Rosmarinus officinalis (leaves and flower), Boswellia serrata (resin), Curcuma longa (rhizome) and Withania somnifera (root) mixed with Sesamum indicum (seed oil) and beehive-wax (Table 3) . The present study has an evidence that declining CRP, CK-MM and Aldo A levels after 6-week specialized topical treatment in combined-sex, female-only and male-only subjects due to decreasing activity of tumor necrosis factor-alpha (TNF-α) while increasing activity of ant-inflammatory interleukins (specially IL-6 and IL-10) by these phytomedicines in the repair mechanisms process .
However, researchers have studied with single plant species and/ or two species in combinations for OA therapy but present study was done by using seven plant species and their different parts along with seed oil of one plant and beehive-wax and results shown may be synergistic effect on biochemical mode of action . The topical phytotherapy in OADs majorly found in animal studies but present study can be an achievement to repair muscles and bones and also skeletal muscles of human by the assessment of normalization of biochemical markers such CRP, CK-MM and AldoA in OADs patients.
Form the present results, it is firmly confirmed that all the biochemical parameters such as CRP, CK-MM and Aldo A were diminished substantially with highly significant values (p<0.0001) in all cases and the percentage of improvements were also highly significant (p<0.0001) in each case after the 6-week of phytotherapy treatment (Table 5) (Figures 4A-4C). As shown in Tables 5 and 6 and Figures 4A-4C, the risk factors for muscular degeneration, skeletal muscle damage and inflammation are closely related. Furthermore, a continuous increasing trend was observed for all three biomarkers regardless of whether the ranges were lower or higher. However, the risk associated with CK-MM levels was substantially higher than that associated with the other two markers such as Aldo A and CRP in subjects with OADs ( Figures 4A-4C).
It is further confirmed that the high and low ranges of all biomarkers such as CRP (<6 mg/L and >6 mg/L), CK-MM (<168 U/L and >168 U/L) and Aldo A (<7.6 U/L and >7.6 U/L) were diminished substantially for combined-sex, female-only and maleonly patients with highly significant values (p<0.0001) in all cases (Table 7) and their percentage of improvements were also highly significant (p<0.0001) in each case ( Figures 4A-4C) after the 6-week of phytotherapy treatment. It is interesting to note that the number of patients and their percentage were not uniform so far as their high and low ranges of biochemical markers were concerned ( Table 6). Table 7 and Figures 4A-4C, the risk factors for muscular degeneration, skeletal muscle damage and inflammation are closely related. Furthermore, a continuous increasing trend was observed for all three biomarkers regardless of whether the ranges were lower or higher. However, the risk associated with CK-MM levels was substantially higher than that associated with the other two markers such as Aldo A and CRP in subjects with OADs. Moreover, the result shows the high predictive values of the three biomarkers in patients with OADs when the correlation coefficients (r-values) are separately calculated among the two biomarkers with highly significant values (p<0.05) in all cases ( Figures 5A-5F).

As shown in
Besides biochemical markers, it has been emphasized several guidelines on different parameters such as pain, daily activities performance and quality of life under KOOS and K-L radiological grading scale of international Literature, the present study dealt with important guidelines as per the standard clinical outcomes for pain and related parameters such as VAS, WOMAC Index, KOOS, KPS and the K-L grading scale used for assessing the effect of novel ISSN: 2334-2846 treatments on OADs by several researchers that pain parameters, functional disability, quality of life by knowing scoring methods and their decreasing data along with biochemical and imaging markers are evaluated along with normalization of elevated levels of the biochemical markers such as CRP, CK-MM, and AldoA by topical application of phytoconstituents correlated with pain scales and radiological gradation through images (Figures 3-7) [ [65][66][67][68].
The present study suggests that phytomedicines contain in phytoextracts have potential therapeutic impact on biochemical mechanism of action in the target cell(s) after topical application of above-mentioned phytomedicines. Moreover, the main aims of the treatment are to control pain, to maintain joint flexibility, to optimize joint and limb function and to improve quality of life, which are archived at the end of 6-week treatment supported by improved radiological features (Figures 2A-2B).

Conclusion
The present study with decreasing levels of biochemical parameters such as CRP, CK-MM and AldoA as a novel approach by the synergistic effect of phytomedicines contain in phytoextracts and can be an alternative of synthetic medicines to prevent OADs permanently. In general, OAD prevents only by using NSAIDs for pain relief or hyaluronic acid and corticosteroid injection for pain, stiffness and inflammation control temporarily and ultimately, total knee replacement (TKR) but these phytomedicines contain in phytoextracts may have ability to repair biochemically in the target cells permanently [70][71][72]. In recent research, phytotherapy indicated no need of revision of TKR [72]. Interestingly, as per international Literature, the present study revealed that pain parameters, functional disability, quality of life by using scoring methods and their decreasing data along with biochemical and imaging markers are firmly concluded that the elevated levels of the biochemical markers such as CRP, CK-MM, and AldoA represent the risks of inflammation, muscle degeneration, and skeletal muscle damage during OAD, can be successfully normalized by topical application of phytoconstituents correlated with pain scales and radiological gradation through images (Figures 3-7). It is suggesting that in future study to know biochemical mechanisms in different cell types such as chondriocytes and osteocytes regeneration with the help of phytochemicals extracted from various Indian medicinal plants.