INTRODUCTION:

Inflammatory arthritis (IA) is a chronic inflammatory systemic disorder that primarily affects smaller joints characterized by periods of severe pain, stiffness, and swelling and destruction of the bones and cartilage^1,2. Steroidal and non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used for the management of inflammatory conditions such as rheumatoid arthritis and other infectious diseases. They reportedly bind to plasma albumin, preventing or inhibiting the thermal denaturation of albumin.

However, they often have toxic or secondary adverse effects resulting from prolonged use which cause damage to the liver, gastrointestinal tract as well as cardiovascular and renal failure,³ The need therefore exists, to explore alternative sources of anti-inflammatory drugs from plant origins. The perceived efficacy, low incidence of serious side effects or relative safety, compared to the synthetic alternatives, as well as the affordability of plant-derived drugs make this search worthwhile. In addition, the ethno-pharmacological uses of many medicinal plants extensively as crude extracts or as pure compounds, have generated considerable interest as it relates to the treatment of various medical conditions including chronic inflammatory diseases. With more than 80 % of the world’s population currently relying on plant-derived medicines for their primary healthcare needs, screening of these plants for potential anti-inflammatory compounds could be a step toward the discovery of safer and more effective compounds⁴.

Phytomedicines and chemical entities derived from plant sources have drawn global attention for their involvement in the treatment of various crippling disorders. One such compound is curcumin, which is the main active ingredient of Curcuma longa (turmeric) used as an additive and condiment in preparing Indian food since time immemorial. These extracts from the roots of Curcuma longa have shown potential anti-inflammatory, antioxidant, chemopreventive, and chemotherapeutic activity in various models of cultured cells and animal studies⁵.

Recently, curcumin has also been found to ameliorate a wide number of clinical conditions including cancer, inflammatory conditions, and digestive disorders. Surprisingly, there are only few scientific reports conducted for understanding the biomedical efficacy of this plant extract especially with reference to the treatment and cure of rheumatoid arthritis and similar other joint inflammatory disorders. A few literature argue that curcumin when administered orally reduces its bioavailability significantly⁶. In physiotherapy, the iontophoretic method has been used for enhancing intradermal drug administration which was basically based on the electromagnetic principle which uses direct electrical current.

Protein denaturation has been identified as the cause of inflammation. Indications are that when living tissues are injured, inflammation results. This is characterized by redness, pain, heat, swelling, as well as loss of function in the affected area. Disruption of the electrostatic, hydrogen, hydrophobic and disulphide bonds in the protein structure occurs. In addition, a complex array of enzyme activation, mediator release, cell migration, tissue breakdown and repair, occur, causing the protein to lose its molecular conformation and functions or become denatured. It is therefore deduced that, compounds which are able to prevent these changes and inhibit thermally or heat induced protein denaturation, have potential therapeutic value as anti-inflammatory agents ⁷.

The stratum corneum is the principle barrier for absorption of drugs through the skin and restricts the permeation of hydrophilic, high molecular weight and charged compounds into the systemic circulation. However many therapeutically active drug molecules are hydrophilic and possess high molecular weights for example, peptides⁸. Iontophoresis simply defined as the use of small amounts of physiologically acceptable electric current to drive ionic (charged) drugs into the body. It is non-invasive technique which uses mild electric current to enhance and facilitate transdermal delivery of variety of drugs. The drug is driven into the skin by electrostatic repulsion⁹, by using the electrode of same polarity as the charge on the drug. Besides the benefits of bypassing the hepatic first pass metabolism and better patient compliance, it has some additional advantages as, delivery of ionized and unionized drugs, enabling continuous or pulsatile delivery of drug, permitting easier termination of drug delivery, restoration of the skin barrier function without producing severe skin irritation, improving the delivery of polar molecules as well as high molecular weight compounds, ability to be used for systemic delivery or local (topical) delivery of drugs, offering better control over amount of drug delivered and reducing considerably inter-individual and intra-individual variability since the rate of drug delivery is more dependent on applied current than on stratum corneum characteristics. Thus, because of many advantages associated with this system, it has been area of growing interest in the local and systemic delivery of many drugs.

Previous study, proposed the stabilization of heat treated BSA by NSAIDs as an assay for replacing animals in the early stages of screening for non-steroidal anti-inflammatory drugs. The present study was undertaken to explore the possible anti-inflammatory and antiarthritic activity of the nanocurcumin and its active ingredient using iontophoresis on certain arthritic patients.

MATERIALS AND METHODS:

Inclusion and exclusion criteria:

In a randomized clinical trial 10 participants with mild to severe stage of rheumatoid arthritis confirmed by radiological and laboratory studies were randomly selected from physiotherapy clinic sivakasi a 8 month period from april 2020 to November 2020

Patients with osteoarthritis , history of fracture , surgery thrombosis of limbs , people with neuropathy , any contraindication or precaution for use of iontophoresis were excluded . the samples were randomly selected and 10 patients were asked about the duration of treatment and duration of follow up. The test is performed.

MATERIALS:

Preparation of Nanocurcumin:

Nanoparticles of curcumin (nanocurcumin) were prepared by a process based on a wet-milling technique.

In vitro anti inflammatory test:

Bovine Serum Albumin Assay (BSA):

The anti-inflammatory activities of the nanocurcumin extracts were determined using a modified version of the BSA assay reported by Williams et al. BSA solution (0.4%, w/v) was prepared in Tris Buffered Saline (one tablet is dissolved in 15 mL of deionized water to yield 0.05M Tris and 0.15M sodium chloride, pH 7.6 at 25⁰C). The pH was adjusted to 6.4 with glacial acetic acid. Stock solutions of nanocurcumin was prepared in water at a concentration of 50 μg/mL or 0.005%, w/v. Respective aliquots of 5.0 µL, 10 µL and 20 µL representing concentrations of 0.25 µg/mL, 0.50 µg/mL and 1.00 µg/mL of the stock solutions were added to test tubes containing 1 mL of 0.4%, w/v BSA buffer solution. The solutions were then heated in a water bath at 72 °C for 10 minutes, and cooled for 20 minutes under laboratory conditions. The turbidity of the solutions (level of protein precipitation) was measured at 660 nm in a Hach Spectrophotometer using an air blank. The experiments were conducted in duplicate and the mean absorbance values were recorded. The percentage inhibition of precipitation (protein denaturation) was determined on a percentage basis, relative to the negative control using the following equation:

Absorbance of control – Absorbance of sample

*% Anti-Denaturation = ------------------------------------------------ x 100

Activity Absorbance of control

*% Anti-Denaturation Activity = % Inhibition of Protein Denaturation = % Anti-inflammatory Activity

Curcumin Iontophoresis procedure:

Preparation of patient:

Position the patient in such a way that is comfortable to the patient. Part to be treated must be exposed and remove dust particles, sebum or sweat to reduce the skin resistance. An electrode patch containing the curcumin drug is placed on skin and this acts as the working electrode another electode is placed elsewhere to complete the electrical circuit and a small current of 0.5 mA/cm2 is applied to deliver the drug through the skin.

RESULTS:

Unlike curcumin, nanocurcumin was found to be freely dispersible in water in the absence of any surfactants. The chemical structure of nanocurcumin was the same as that of curcumin, and there was no modification during nanoparticle preparation.

Anti inflammatory test:

Screened for anti inflammation activity using the BSA protein denaturation assay, 20mg/l of nanocurcumin displayed greater than 69% inhibition of protein denaturation. 50 mg/l showed 100% inhibition. [Figure: 1]

Figure: 1 In vitro anti inflammation assay:

Notably, although 69% inhibition of protein denaturation represents the minimum limit for potential anti-inflammatory agents, Dosage which showed a 100% antidenaturation activity were selected for further investigation, in order to increase the possibility of identifying potential lead anti-inflammatory compounds. [Figure:2]

Figure: 2 Anti inflammatory tests result:

Iontophoresis Studies in Human Subjects:

All ten participants tolerated the iontophoresis treatments well and no sign of irritation or erythema was observed on the skin at the time when the patches were removed. None of the participants reported any problems following the experiments due to the iontophoresis treatment, although they were encouraged to do so when signing the consent.

The drug nanocurcumin should be storable in liquid or dry form in the patch and should be stable. It should be soluble in aqueous media and be charged. [Figure: 2,3] The isoelectric point should be in the range of smaller than 4 or greater than 7.4. The iontophoretic device should deliver the drug in following manner 20-50mg drug/day of molecular weight of 300 Da, 2-5mg drug/day of molecular weight of 1000 Da and 100μg drug/day of molecular weight of 5000Da.

Figure: 2 Clinical set up for Iontophoresis:

Figure: 3 Human trial

The nonparametric Mann–Whitney Utest used to compare iontophoresis and passive sites conﬁrmed that iontophoresis signiﬁcantly increases the delivery of curcumin to dermis (p<0.0001) compared with passive application of curcumin.

DISCUSSION:

An association between weight loss and inflammatory joint disease has been recognized as early as in the 1940s when Hornell gave a detailed description of RA where inflammation was one of the key significant features ¹⁰. In the present study, the anti-inflammatory and antiarthritic effects of nanocurcumin extracts using iontophoresis as a treatment method in arthritis patients were studied. During the analysis of the extract, a higher content of curcumin, i.e., about 50mg/l, was found effective. This study demonstrated that cathodal iontophoresis delivers significantly more quantifiable amounts of nanocurcumin across human skin.

In this study, Adoption of the BSA protein denaturation assay for the in vitro evaluation of anti-inflammatory potential of nanocurcumin extracts circumvented the ethical issues associated with the use of humans, especially in the early stages of screening for nanocurcumin with potential lead anti-inflammatory compounds. In addition, protein denaturation has been described as a pathological process which involves the loss of configuration, and as a result, loss of functionality ¹¹. This makes the reduction in protein denaturation, and by extension the BSA protein denaturation assay, ideal for the determination of anti-inflammatory potential. It should be noted that the experiments were conducted at pH 6.4, which represents the pathological pH (6.2 - 6.5) at which, reportedly heat treated BSA is stabilized (denaturation is inhibited) by nanocurcumin.

Like wise Mukund Nagarnaik et al., says that, the histopathological assessment revealed that the uniform and compactly arranged normal synovial membrane was damaged and deranged with significant depletion of synoviocytes. Group of rats receiving oral curcumin along with iontophoretic application showed the best recovery and restoration of the ankle joint. The synovial architecture of the ankle joint of the two groups, viz, oral curcumin with and without plain topica application, showed recovery from damage; however, complete restoration was not observed in either of them. The effective treatment among the animals receiving iontophoretic application of curcumin might be due to thereduced skin resistance, which is brought about by the electric potential gradient of the iontophoresis technique^12,13.The reduced skin resistance allows better penetration and, hence, increases the bioavailability. This in turn probably would have elucidated antiarthritic and anti-inflammatory actions on the antigens.

CONCLUSION:

The outcomes of the presented study suggest that transdermal iontophoretic delivery may be a valuable tool for drug delivery in artheritis patients. The incompliance of the these patients to the medications is well known; hence, this study opines that simultaneous use of transdermal nanocurcumin via iontophoretic application is very effective in countering the arthritic symptoms. Nevertheless, further studies with larger sample size are recommended to understand the manifestations better and exploit the concept for clinical implications.

DATA AVAILABILITY:

The data used to support the findings of this study are available from the corresponding author upon request.

CONFLICTS OF INTEREST:

The authors declare that they have no conflicts of interest.

ACKNOWLEDGMENTS:

The authors are grateful to the Ponmuthu Physiotherapy clinic in Madurai and Saveetha College of Physiotherapy for their guidance.

REFERENCES:

1. F. Cavaleri, “Presenting a new standard drug model for turmeric and its prized extract, curcumin,” International Journal of Inflammation, vol. 2018, Article ID 5023429, pages, 2018, 118-132

2. Anubhav Das, Anitha Roy, S. Rajeshkumar, T. Lakshmi. Anti-inflammatory Activity of Turmeric Oil Mediated Silver Nanoparticles. Research J. Pharm. and Tech. 2019; 12(7):3507-3510.

3. Nirisha Sriram, Gheena. S, Samrithi Yuvaraj. Effects of turmeric on oral submucous fibrosis: A Systematic Review. Research J. Pharm. and Tech. 2015; 8(8):1051-1055.

4. Suman Saha, Amit Roy, Sanjib Bahadur, Ananta Choudhury. Bioenhamcement of Curcumin by Dual approach. Research J. Pharm. and Tech. 2016; 9(8):1059-1063.

5. MD Wandhare, UA Deokate, SS Khadabadi, SP Hadke, HA Sawarkar. Comparative Estimation of Curcumin Content from Marketed Herbal Anti Rheumatic Tablets Formulation. Asian J. Research Chem. 2009; 2(3): 340-343.

6. Ruchika Sharma, Neha Kumari, M.S Ashawat, C.P.S Verma. Standardization and Phytochemical Screening analysis for Herbal Extracts: Zingiber officinalis, Rosc., Curcuma longa Linn., Cinnamonum zeylanicum Nees., Piper longum, Linn., Boerhaavia diffussa Linn. Asian J. Pharm. Tech. 2020; 10(3):127-133.

7. Sweetha G., Sangeetha B., Prabhu S.. A Review on Curcumin Nanoparticles and Its Controlled Delivery to Treat Degenerative Diseases. Asian J. Pharm. Tech. 2013; Vol. 3: Issue 4, Pg 218-222.

8. Sabale Prafulla, Potey Lata, Rahangdale Priya, Sabale Vidya. Novel Curcumin Derivatives: Targeted for Anti-Inflammatory Activity. Asian J. Research Chem. 2019; 12(2):49-54.

9. LatheeshjlalL, Sunil Murala, Vaidya Mehul J, G Swetha, Phanitejaswini Swapna. Bioavailability Enhancement of Curcumin through Mucoadhesive Drug Delivery System. Research J. Pharm. and Tech. 2011; 4(3): 457-460.

10. Liu, H. Lou, L. Zhao, and P. Fan, “Validated LC/MS/MS assay for curcumin and tetrahydrocurcumin in rat plasma and application to pharmacokinetic study of phospholipid complex of curcumin,” Journal of Pharmaceutical and Biomedical Analysis, 2006; 40(3): 720–72.

11. S. Blaise, M. Roustit, C. Millet, C. Ribuot, J. Boutonnat, and J. Cracowski, “Cathodal iontophoresis of treprostinil and iloprost induces a sustained increase in cutaneous flux in rats,” British Journal of Pharmacology. 2011; 162(3): 557–565,.

12. Mukund Nagarnaik, Arun Sarjoshi, Ajay Bodkhe, Bhupendra Khanal, Mayuri Pise, Girish Pandya. Characterization of active constituents in Turmeric powder and validation of method for curcumin in samples. Asian J. Research Chem. 2015; 8(10): 643-64

13. P. Venugopalan, T. V. Deepthi. Chemical and Pharmacological Studies on Curcuminoids. Asian J. Research Chem. 2014; 7(3): 355-365.

Received on 30.11.2020 Modified on 19.03.2021

Research J. Pharm.and Tech 2022; 15(2):825-829.

DOI: 10.52711/0974-360X.2022.00137