Preparation, Characterization and Pharmacological evaluation of

Bauhinia variegata Lauha Bhasma

 

Rupali Ajesh Gulalkari

Dept of Chemistry, BJS’S ASC College wagholi Pune – 07.

 

ABSTRACT:

The study was carried out to prepare, characterize, and investigates the pharmacological evaluation of Bauhinia variegata lauha bhasma (BVLB). The Lauha bhasma (LB) was prepared according to the reported standard method that involved the destruction of metallic state followed by conversion of crude product into bhasma state. The purification was done using Triphala extract prepared in cow-urine. The destruction of the metallic test was carried out using process juice of BV. At the last, the preparation of BVLB is subjected to a closed crucible system to get the final product. The characterization of BVLB was done by some preliminary examination followed by specific methods viz. X-ray diffractometry (XRD), Infrared (IR) spectroscopy, and UV-visible spectrophotometry. The In-vitro anti-inflammatory, anti-arthritic and antioxidant activity was carried out for BVLB at different concentrations of 50, 100, 200, and 250μg/mL. The said In-vitro activities were screened using percent inhibition of heat-induced hemolysis, the percent inhibition of protein denaturation, proteinase inhibition, and DPPH free radical scavenging activity. BVLB preliminary investigation showed 1.53 % aqueous and 3.95 % HCL solubility whereas specific characterization methods showed the formation of micro and nanocrystals of F2CO3. The BVLB showed % inhibition of hemolysis of in between 5.26 ± 0.95 to 31.20±4.12 whereas percent inhibition of protein denaturation in between 12 ± 1.22 to 52.22 ± 4.69. The percent proteinase inhibition of BVLB at a different concentration from 50, 100, 200, and 250μg/mL was estimated and found in between 3.26 ± 0.86 to 51.22 ± 3.12. The free radical scavenging activity of BVLB was found from 10.11 1 ± 0.89 to 52.05 ± 2.05. From the current investigation, it can be conducted that BVLB has significant In-vitro anti-inflammatory, anti-arthritic and antioxidant activity. The formed nanocrystals and presence of BV active constituents would be the underlying intermediaries for significant activity.

 

 

INTRODUCTION:

Indian Ayurvedic system of medicine has tremendous potential to treat several clinical aliments. The scientists are much interested and keen to explore the medicinal value of plant based medicines. Amongst some potent plants Bauhinia variegata (BV) Linn. (Leguminosae) is an essential component of several ayurvedic preparation used for several clinical treatments¹. Bauhinia variegata L have a broad spectrum of activities such as antibacterial, anticancer and antioxidant activity of bark^1,2.

 

Hepatoprotective, antioxidant and anti-hyperlipidemic activity reported using the bark preparation^3,4. Antitumor activity was screened against dalton's ascitic lymphoma⁵, Novel flavonol and glycosides isolated form Bauhinia variegata Linn showed significant anti-inflammatory activity^6,7. Significant immunomodulatory⁸, anti-ulcer⁹ and anti-nephrotoxic activity¹⁰ of Bauhinia variegata Linn. was screened using In-vivo and In-vitro pharmacological models. Bauhinia variegata Linn is known as “kanchanara” in Ayurveda and one of the commonly used folk medicine in India. In Ayurveda LB have important consideration in treatment of several acute and chronic blood related diseases conditions. It is a complex of herbal minerals extensively used as hematinic agent. Along with that is has a potential curative effect against treatment of chronic fever, breathlessness, for enhancing strength and anti-aging properties¹¹. Combination of LB along with BV Linn will have additive effect and improved outcomes in significant way. The modern medicines are effective to counter the acute diseases and treatment approaches are symptomatic, however chronic treatment with allopathic medicines in conditions like diseases like arthritis pose for severe toxic effects. It was advocated by several studies that chronic consumption of NSAID will leads to hepatotoxicity, nephrotoxicity, disturbed acid-base balance, ulceration and bleeding¹². Immunosuppressants like corticosteroid are commonly prescribed in inflammatory conditions. But chronic consumptions leads to acquisition of infections, disturbed hormonal balance, weight gain etc¹³. Disturbed endogeneous anti-oxidant mechanism and uncontrolled free radical generation is an underlying cause for several life threatening diseases like diabetes, arthritis, cancer, and organ damage and diseases related to nervous system^14-18. Considering the benefits of BVLB following study was designed to prepare, characterize and screen for pharmacological activities of the same.

 

MATERIAL AND METHODS:

Raw material and chemical agents used:

The raw material was selected based upon the studies previously mentioned the same. This includes pure iron powder or thin iron sheets, magnetite or naturally occurring Fe3O4, rusted iron (known as mandur), Iron pyrites (FeS2 ) (known as makshika) and naturally occurring  FeSO4  (kasis). The chemical used in the different assay methods were procured from the institutional stores. All the agents used were AR grade with the highest purity.

 

Preparation and purification of BVLB:

The reported standard methods were adapted for the preparation of LB. The preparation of LB is carried out primarily in three steps which involve purification of iron by Ayurvedic process, destruction of metallic state followed by conversion of crude product into bhasma state. The purification process was carried out using Triphala extract prepared in cow-urine. The obtained iron powder was heated to red heat and then dipped again in Triphala extract successively seven times. The destruction of the metallic test was carried out using the process of “marana” in the juice of BV (Kanchnar). Which involves the purified iron powder was mixed with BV plant juice in a mortar. Further, the mixture was triturated till to get a homogeneous paste. The last step in the preparation of BVLB involved subjecting the formed powder into the closed crucible system to get the LB¹⁸.

 

Characterization of BVLB:

The prepared BVLB was subjected to some preliminary examination which includes solid-state, colors, solubility in aqueous, organic solvents, and dilutes HCL. The specific characterization of BVLB was carried out using X-ray diffractometry, Infrared spectroscopy, and UV-visible spectrophotometry. For R-RD pattern of BVLB powdered samples was spread as a thin layer on cavity mounts and recorded on Phillips X pert pro powder diffractometer in the diffraction angle range (10-90) 2θ. The crystalline size of BVLB was calculated using the Debye-Scherrer equation. Solid-state IR spectra BVLB were recorded in KBr pellets using FTIR Perkin-Elmer infrared spectrophotometer in the normal region (4000-450) cm-1. For UV-Visible 1.00 mg of BVLB was taken and observations were recorded in the region (400-100) nm on a Shimadzu UV spectrometer model against pure KBr pellets as the blank.

 

Pharmacological investigation of BVLB:

Preparation of sample and erythrocyte suspension:

BVLB was prepared by dispersing in distilled water. The different concentrations ranging from50 μg/ml to 250μg/ml were prepared and taken for the assay purposes. For screening of anti-inflammatory activity, the erythrocyte suspension was prepared according to the methods reported previously with slight modifications ^19,20.  The whole blood sample of about 2.5ml was taken from the healthy human volunteer who has not taken any NSAIDs for the past 15 days and did not have any inflammatory symptoms. The collected blood was subjected to centrifugation for 5 minutes at 3000rpm in presence of heparin. The supernatant was removed and tube content was washed two to three times with an equal volume of normal saline solution (0.9% NaCl). The packed blood cell was suspended 10% (v/v) in an isotonic buffer (10 mM sodium phosphate) solution of pH 7.4

 

Heat-Induced Hemolysis:

The heat-induced hemolysis of erythrocyte assay was carried out by using reported methods with slight modification ^19,20,21. The method involves 50μl of blood cell suspension, 50μl of test sample mixed with 2.9 mL phosphate buffer (pH 7.4). The formed mixture was incubated at 540C (the temperature was controlled using a thermostat with ±0.1° C) for 25 min in a shaking water bath followed by centrifuging at 2500 rpm for 3minutes). The supernatant was separated and absorbance was measured at 540nm using a Shimadzu UV-2600i spectrophotometer. The concentration viz. 50, 100, 200, and 250 mcg/mL of BVLB were used to study the effect on heat-induced hemolysis 19. The degree of hemolysis was calculated using the following equation

 

% inhibition of hemolysis = 100 X (A1 - A2/A1)

 

Where A1 = absorption of the control, and A2 = absorption of the test sample mixture.

 

Effect on Protein Denaturation:

Protein denaturation assay was carried out according to the method described earlier with slight modification ^21,22. The assay mixture (3mL) consisted of 20μl of 1% bovine albumin, 2.8 mL of phosphate-buffered saline (pH 6.4), and different concentration of BVLB (50, 100, 200, and 250 mcg /mL). The assay mixture was mixed well and incubated for 20 minutes at 37⁰C. Further, the reaction mixture was heated at 70C for 5 min. After getting the reaction mixture at room temperature the degree of protein denaturation incubated as turbidity was measured at 660 nm using a Shimadzu UV-2600i spectrophotometer. The Phosphate buffer solution served as the control. The percentage inhibition of protein denaturation was calculated by using the following formula:

 

% inhibition of denaturation = 100 X (A1 - A2/A1)

 

Where A1 = absorption of the control sample,

and A2 = absorption of the test sample.

 

Proteinase Inhibitory Activity:

Proteinase inhibitory activity was carried out according to the method described earlier with slight modification^21,22,23. The assay mixture 2 mL contains 0.06 mg trypsin, 1mL of 20 mM Tris-HCl buffer (pH 7.4), and 1 mL test sample (50 μl of  BVLB and 0.95mL methanol). The reaction mixture was incubated for 5 minute at 37⁰C followed by addition of 1 mL of 0.8% (w/v) casein. Further the assay mixture was incubated for 20 minute for same temperature. The reaction was terminated with addition of 2 mL of 70% perchloric acid. The reaction mixture was subjected to centrifugation for 10 minute at 2500rpm and supernatant was separated. The absorbance was measured at 210 nm against buffer as the blank. Phosphate buffer solution served as the control. The percentage inhibition of protein denaturation was calculated by using the following formula:

 

% inhibition of denaturation = 100 X (A1 - A2/A1),

 

Where A1 = absorption of the control sample, and A2 = absorption of the test sample.

 

DPPH Free radical scavenging activity:

The free radical scavenging activity of BVLB was carried out according to the previously reported methods with slight modification^24,25,26. The stable 1,1-diphenyl-2- picrylhydrazyl (DPPH) free radical assay involves a reaction mixture of BVLB (50, 100, 200, and 250 μg /mL) added in 3ml of a 0.004% methanol solution of DPPH. The change in the absorbance at 517nm was measured after 30 min. The percent DPPH scavenging activity was carried out using the following formula and the inhibition curves were prepared and IC50 values were obtained. The standard antioxidant used was α-tocopherol (α-Toco)

% DPPH scavenging = 100 X (1 - A2/A1),

 

Where A1 = absorption of the control sample, and A2 = absorption of the test sample/Standard.

 

RESULTS:

Preliminary examination of BVLB:

The state of BVLB was found solid with dark brown coloration. The aqueous solubility of BVLB was carried out for the sample weight of 1gm and added in 10 mL of distilled water and subjected for boiling. After 15 minutes the mixture was filtered and the water solubility was found as 1.53 %. The acid solubility in HCl (0.3 %) was also determined in the same way and found at 3.95 %. The solubility in common organic solvents like methanol, ethanol, acetone, chloroform, n-naphthalene, benzene was and found practically insoluble.

 

Figure 1. Crystallographic parameters from XRD patterns of BVLB

 

X-ray diffractometry:

The XRD is useful to examine the different crystal modifications of iron oxide and understand the medicinal properties and of LB against pure iron oxide. The XRD patterns with relevant details are illustrated in figure the summarized results and conclusion of XRD investigations are given in Table 1.

Table 1 Crystallographic parameters from XRD patterns of BVLB

Nature and Crystal System from XRD	Major constituents	Crystallite size	Space group	Space group	Space group	Space group
Microcrystalline; Rhombohedral	Hamatite	039.7 nm	R-3C	R-3C	R-3C	R-3C

 

From the examination of XRD patterns and the data obtained by the analysis of XRD and crystallite size determinations, it can be reported that the BVLB is a microcrystalline and nanocrystalline solid. XRD lines are not distinct and sharp due to mixtures of heterogeneous particles of different crystallite sizes and predominantly of iron oxides (Fe2O3). The crystal symmetry of Fe2O3 is rhombohedral.

 

Infrared spectroscopy:

There significant peaks in the IR spectra obtained for BVLB was showed in figure 2 and table 2. The most important and characteristic peak is in the region (1205-1215) Cm-1 was observed known to represent C-O. However, such peaks are absent in the standard IR spectra of pure Fe203 hence, BVLB carries a significant difference in potential investigation The second peak recorded in the region (765-785) Cm-1 is not resolved at all, and therefore, it is difficult to make any assignment. The third distinct peak in the region (500-510) Cm-1 is also characteristic because its position and intensity are similar. In literature, this peak is assigned to Fe2O3 and its identity is confirmed in infrared spectra of   Fe2O3. Therefore, in structural analysis of BVLB, which is composed of Fe2O3 predominantly, this peak may be considered as a diagnostic peak for BVLB.

 

Table 2. Significant IR frequencies Cm^-1 and their assignment for BVLB

 

Figure 2. Significant IR frequencies Cm^-1 and their assignment for BVLB

 

UV-visible spectrophotometry:

UV visible spectrophotometry was rarely attempted for the characterization of solid-state compounds. We have adapted the KBr as it's been recorded successfully and the same transparent- pellets are found to be useful for UV spectra. Significant absorption was observed in the ultraviolet region (250-360 and visible region (390-480) nm for BVLB. Both these peaks are attributable to π - π* electronic transitions and the nature and presence of these peaks provide additional support in favor of incorporation of organic components in BVLB, through the process. The origin of this π - π* transitions will be understood only when the exact solid-state structure of the LB will be elucidated.

 

Table 3. Significant absorption maxima (λ max) of BVLB

 

 

Figure 3. Significant absorption maxima (λ max) of BVLB

 

Effect of BVLB on heat-induced hemolysis:

The effect of BVLB percent inhibition of heat-induced hemolysis of erythrocyte at a different concentration from 50, 100, 200, and 250μg/mL is shown in figure 4a. BVLB showed percent inhibition of hemolysis in a dose-dependent manner.  Inhibition % of hemolysis from BVLB was within the range from 5.26 ± 0.95 to 31.20±4.12. The BVLB at 250μg/mL showed maximum percent inhibition of heat-induced hemolysis of erythrocytes.

 

Effect of BVLB on percent proteinase inhibition:

The effect of BVLB on percent proteinase inhibition is shown in figure 4b. The BVLB can inhibit proteinase inhibition in a dose-dependent manner. The percent proteinase inhibition of BVLB at a different concentration from 50, 100, 200, and 250μg/mL was estimated and found in between 3.26 ± 0.86 to 51.22 ± 3.12. The BVLB at 250μg/mL showed maximum percent inhibition of protein denaturation.

 

Effect of BVLB on percent inhibition of protein denaturation:

The effect of BVLB on the percent inhibition of protein denaturation was shown in figure 4c. The BVLB is able to inhibit percent protein denaturation in a dose-dependent manner. The percent protein denaturation of BVLB at a different concentration from 50, 100, 200, and 250μg/mL was estimated and found in between 12 ± 1.22 to 52.22 ± 4.69. The BVLB at 250μg/mL showed maximum percent inhibition of protein denaturation.

 

Values are expressed as mean ± SD, n=3, IC₅₀ for % inhibition of hemolysis was found 400 μg/mL; IC₅₀ for % proteinase inhibition was found  2.44.04 μg/mL; IC₅₀ for % inhibition of protein denaturation  was found  239.37 μg/mL; IC₅₀ for DPPH scavenging activity was found 240.3 μg/mL

 

 

Effect of BVLB on DPPH Free radical scavenging:

The effect of BVLB on free radical scavenging activity is shown in figure 4d. The free radical scavenging activity of BVLB and α-tocopherol (standard) at a different concentration from 50, 100, 200, and 250μg/mL was estimated and found in between 10.11 1 ± 0.89 to 52.05 ± 2.05 and 25.15 ±1.20 to 83.12 ± 2.45 respectively The BVLB showed dose-dependent free radical scavenging activity.

 

DISCUSSION:

LB is one of the ancient preparations commonly used as folk medicines with a combination of several plant juices or decoction. The Ayurvedic medicinal system uses metals and metallic preparations that are unique and known as the Indian subcontinent recommended for the treatment of chronic ailments¹⁸.  A Bhasma stands for ash derived from incineration where raw materials are subjected for processes like purification, metallic taste masking, and combining with active plant constituents. Bhasma mainly contains Fe, Cu, S have a particle size (1-2 μ) which facilitates absorption and bears the characteristics of nanomedicines^18,27. LB is recommended for different ailments like anemia, hemorrhoids, bleeding disorders, the pain of different origins, fevers, diseases of liver and spleen, dropsy, rheumatic arthritis, gout, skin disorders, obesity, cough, and dyspnea, etc ²⁸. In consideration of the potential of LB we have combined it with the plant juice of BV as an additive significant active constituent. The characterization studies showed the fine microcrystals rhombohedral and nano-crystals of BVLB, Fe present in the elemental form as hematite. To explore the potential benefits of several compounds various in-vitro anti-inflammatory, anti-arthritis, and antioxidant methods are reported ^29,30. In the inflammatory response, the leukocytes plays important role in the cellular infiltration. As a cascade of inflammation leukocytes releases lysosomal enzymes which cause tissue damage and further progression inflammation. Erythrocytes membranes are similar to that of lysosomal membrane; inhibition of erythrocyte hemolysis may offer insights into the inflammatory process ^31,32,33. Percent inhibition and stabilization of erythrocytes cell membranes may inhibit or delayed the lysis and subsequent release of the cytoplasmic contents. Which is further responsible to minimize the tissue damage and confirms the probable anti-inflammatory effect. The current investigation showed that BVLB has the potential to stabilize the cell membrane and inhibits hemolysis which confirms its anti-inflammatory activity. Several studies reported that the denaturation of protein molecules is one of the common cascades in arthritic inflammation^20,21,34. One of the principal mechanisms of action of NSAIDs is the protection against protein denaturation as reported in some of the earliest studies ^35,36. Inhibition of protein denaturation may be an important strategy to prove the anti-arthritic activity of an investigational compound. Along with that proteinases have been reported they are a significant contribution to the development of arthritic conditions. The neutrophils contain several serine proteinases in their lysosomal granules and are responsible for inflammatory processes. Studies advocated that inhibition of proteinases may delay the inflammation and progression of join degradation ^37,38. Elaborated generations of free radicals have been reported in several studies and their pathophysiological role has been explored. The disturbed endogenous anti-oxidant mechanism is mainly responsible for the development of numerous clinical conditions. To explore the potential anti-oxidant activity of test compound DPPH free radical scavenging activity is commonly used³⁹. Radical scavenging activities are significantly essential to inhibit the injurious starring role of free radicals in the number of diseases. Several studies have advocated that DPPH free radical scavenging is well an established and accepted method for screening of antioxidant activity as it is relatively simple and requires less time. The current study result revealed that the BVLB have free radical scavenging activity when compared to standard anti-oxidant. In the conclusion, it can be stated that the BV along with LB have a respectable in-vitro anti-inflammatory, anti-arthritic, and anti-oxidant ability and can be recommended in inflammatory diseases. Further animal studies are needed to explore and confirm the potential benefits of BVLB.

 

ACKNOWLEDGEMENTS:

I thank to the management and Major Dr Ashok V. Giri principal Bhartiya Jain Sanghatanas Arts, Commerce & Science College – Pune for motivation providing necessary facilities to carry out the work.

 

REFERENCES:

1.     Mishra A, Sharma AK, Kumar S, Saxena AK, Pandey AK. Bauhinia variegata leaf extracts exhibit considerable antibacterial, antioxidant, and anticancer activities. BioMed Research International. 2013 Aug;2013. https://doi.org/10.1155/2013/915436

2.     Rajani GP, Ashok P. In vitro antioxidant and antihyperlipidemic activities of Bauhinia variegata Linn. Indian journal of pharmacology. 2009 Oct;41(5):227. doi: 10.4103/0253-7613.58513

3.     Bodakhe SH, Ram A. Hepatoprotective properties of Bauhinia variegata bark extract. Yakugaku zasshi. 2007 Sep 1; 127(9):1503-7. https://doi.org/10.1248/yakushi.127.1503

4.     Tripathi AK, Gupta PS, Singh SK. Antidiabetic, anti-hyperlipidemic and antioxidant activities of Bauhinia variegata flower extract. Biocatalysis and agricultural biotechnology. 2019 May 1; 19:101142. https://doi.org/10.1016/j.bcab.2019.101142

5.     Rajkapoor B, Jayakar B, Murugesh N. Antitumour activity of Bauhinia variegata on Dalton’s ascitic lymphoma. Journal of Ethnopharmacology. 2003 Nov 1;89(1):107-9. https://doi.org/10.1016/S0378-8741(03)00264-2

6.     Yadava RN, Reddy VM. Anti-inflammatory activity of a novel flavonol glycoside from the Bauhinia variegata Linn. Natural product research. 2003 Jan 1;17(3):165-9.  https://doi.org/10.1080/1478641031000104127

7.     Rao YK, Fang SH, Tzeng YM. Antiinflammatory activities of flavonoids and a triterpene caffeate isolated from Bauhinia variegata. Phytotherapy research. 2008 Jul;22(7):957-62. https://doi.org/10.1002/ptr.2448

8.     Ghaisas MM, Shaikh SA, Deshpande AD. Evaluation of the immunomodulatory activity of ethanolic extract of the stem bark of Bauhinia variegata Linn. International Journal of Green Pharmacy (IJGP). 2009;3(1). DOI: http://dx.doi.org/10.22377/ijgp.v3i1.60

9.     Rajkapoor B, Jayakar B, Anandan R, Kavimani S. Anti-ulcer effect of Bauhinia variegata Linn. in rats. Journal of natural remedies. 2003 Jun 1; 3(2):215-7. https://doi.org/10.18311/jnr/2003/170

10.  Sharma RK, Rajani GP, Sharma V, Komala N. Effect of ethanolic and aqueous extracts of Bauhinia variegata Linn. on gentamicin-induced nephrotoxicity in rats. Ind J Pharm Edu Res. 2011 Apr 1;45(2):192-8.

11.  Joshi N, Dash MK, Dwivedi L, Khilnani GD. Toxicity study of Lauha Bhasma (calcined iron) in albino rats. Ancient science of life. 2016 Jan;35(3):159. doi: 10.4103/0257-7941.179870

12.  James DS. The multisystem adverse effects of NSAID therapy. Journal of Osteopathic Medicine. 1999 Nov 1;99(11):1-7. https://doi.org/10.7556/jaoa.1999.02

13.  Fireman M, DiMartini AF, Armstrong SC, Cozza KL. Immunosuppressants. Psychosomatics. 2004 Jul 1;45(4):354-60. https://doi.org/10.1176/appi.psy.45.4.354

14.  Mathew BB, Tiwari A, Jatawa SK. Free radicals and antioxidants: A review. Journal of Pharmacy Research. 2011 Dec;4(12):4340-3

15.  Krishna KV, Karuppuraj V, Perumal K. Antioxidant activity and Folic acid content in indigenous isolates of Ganoderma lucidum. Asian Journal of Pharmaceutical Analysis. 2016;6(4):213-5. doi : 10.5958/2231-5675.2016.00032.6

16.  Balaji EV, Selvan A, Srinivasan A, Nandhini S. Punica granatum root (s): Phytocompounds analysis, Anti-oxidant and Anti-microbial activity. Asian Journal of Pharmaceutical Analysis. 2019;9(3):123-7. doi : 10.5958/2231-5675.2019.00023.1

17.  Valli G, Jeyalakshmi M. Preliminary phytochemical and antioxidant study of Odina woodier leaf extract. Asian Journal of Pharmaceutical Research. 2012;2(4):153-5.

18.  Singh TR, Gupta LN, Kumar N. Standard manufacturing procedure of Teekshna lauha bhasma. Journal of Ayurveda and integrative medicine. 2016 Apr 1;7(2):100-8. https://doi.org/10.1016/j.jaim.2015.08.003

19.  Parvin MS, Das N, Jahan N, Akhter MA, Nahar L, Islam ME. Evaluation of in vitro anti-inflammatory and antibacterial potential of Crescentia cujete leaves and stem bark. BMC research notes. 2015 Dec;8(1):1-7. https://doi.org/10.1186/s13104-015-1384-5

20.  Gunathilake KD, Ranaweera KK, Rupasinghe HP. In vitro anti-inflammatory properties of selected green leafy vegetables. Biomedicines. 2018 Dec;6(4):107. https://doi.org/10.3390/biomedicines6040107

21.  Ranasinghe P, Ranasinghe P, Abeysekera WK, Premakumara GS, Perera YS, Gurugama P, Gunatilake SB. In vitro erythrocyte membrane stabilization properties of Carica papaya L. leaf extracts. Pharmacognosy research. 2012 Oct;4(4):196. doi: 10.4103/0974-8490.102261

22.  Okoli CO, Akah PA, Onuoha NJ, Okoye TC, Nwoye AC, Nworu CS. Acanthus montanus: An experimental evaluation of the antimicrobial, anti-inflammatory and immunological properties of a traditional remedy for furuncles. BMC complementary and alternative medicine. 2008 Dec;8(1):1-1. https://doi.org/10.1186/1472-6882-8-27

23.  Elumalai A, Prakash YG. Evaluation of anti-arthritic activity of ethanolic extract of Pisonia grandis R. Br. Asian Journal of Pharmaceutical Research. 2012;2(3):91-3.

24.  Dey A, Kanjilal S, Adhikari A, Bhatt BN, Chakraborty T, Chakraborty P, Gandhi D, Narwaria A, Katiyar CK. Anti-inflammatory activity of Zandu Rhumayog Forte and Rhumasyl Gel in acute and chronic inflammatory models. Annals of Ayurvedic Medicine. 2019;8(3):104-13.

25.  Hardik Joshi, Manoj Pagare, Leena Patil, Vilasrao Kadam. In–Vitro Antioxidant Activity of Ethanolic Extract of Leaves of Buchanania Lanzan Spreng. Research J. Pharm. and Tech. 4(6): June 2011; Page 920-924.

26.  Razi SB, Zaaeri F, Javar HA. An HPLC Method for detection of Anti-inflammatory Drugs in Bone and Cartilage health supplements. Asian Journal of Pharmaceutical Analysis. 2020;10(2):67-76. doi : 10.5958/2231-5675.2020.00012.5

27.  Pal D, Sahu CK, Haldar A. Bhasma: the ancient Indian nanomedicine. Journal of advanced pharmaceutical technology & research. 2014 Jan;5(1):4. doi: 10.4103/2231-4040.126980

28.  Gupta KV, Pallavi G, Patgiri BJ. Critical review on the pharmaceutical vistas of Lauha Kalpas (Iron formulations). Journal of Ayurveda and integrative medicine. 2012 Jan;3(1):21. doi: 10.4103/0975-9476.93944

29.  Djuichou Nguemnang SF, Tsafack EG, Mbiantcha M, Gilbert A, Atsamo AD, Yousseu Nana W, Matah Marthe Mba V, Adjouzem CF. In vitro anti-inflammatory and in vivo antiarthritic activities of aqueous and ethanolic extracts of Dissotis thollonii Cogn.(Melastomataceae) in rats. Evidence-Based Complementary and Alternative Medicine. 2019 Nov 15;2019. https://doi.org/10.1155/2019/3612481

30.  Rai R. Herbal Folk Remedies in cure of Arthritis by Ethnic communities in Madhya Pradesh, India. Asian Journal of Research in Pharmaceutical Science. 2016;6(3):177-84. doi : 10.5958/2231-5659.2016.00024.2

31.  Radi ZA, Kehrli Jr ME, Ackermann MR. Cell adhesion molecules, leukocyte trafficking, and strategies to reduce leukocyte infiltration. Journal of Veterinary Internal Medicine. 2001 Nov;15(6):516-29. https://doi.org/10.1111/j.1939-1676.2001.tb01586.x

32.  Padmanabhan P, Jangle SN. Evaluation of in-vitro anti-inflammatory activity of herbal preparation, a combination of four medicinal plants. International journal of basic and applied medical sciences. 2012;2(1):109-16.

33.  Pagar KR, Khandbahale SV, Kasar PM. Osteoarthritis: Pathophysiology and Current Treatment Modalities. Asian Journal of Pharmaceutical Research. 2019;9(4):289-98. doi : 10.5958/2231-5691.2019.00046.7

34.  Dhalendra G, Satapathy T, Roy A. Animal models for inflammation: A review. Asian Journal of Pharmaceutical Research. 2013;3(4):207-12.

35.  Saso L, Valentini G, Casini ML, Grippa E, Gatto MT, Leone MG, Silvestrini B. Inhibition of heat-induced denaturation of albumin by nonsteroidal antiinflammatory drugs (NSAIDs): Pharmacological implications. Archives of pharmacal research. 2001 Apr;24 (2):150-8. https://doi.org/10.1007/BF02976483

36.  Khan H, Ali M, Ahuja A, Ali J. Validated HPLC-UV Method for Simultaneous Determination of Some Anti-Inflammatory and Analgesic Drugs. Asian Journal of Pharmaceutical Analysis. 2016;6(3):183-7. doi : 10.5958/2231-5675.2016.00027.2

37.  Oikonomopoulou K, Diamandis EP, Hollenberg MD, Chandran V. Proteinases and their receptors in inflammatory arthritis: an overview. Nature Reviews Rheumatology. 2018 Mar;14(3):170-80. https://doi.org/10.1038/nrrheum.2018.17

38.  Anbarasi A, Vidhya R. Evaluation of in vitro anti-inflammatory activity of Tephrosia purpurea (Seed). Asian Journal of Pharmaceutical Research. 2015;5(2):83-9. doi : 10.5958/2231-5691.2015.00012.X

39.  Gangadhara MM, Dinesha R, Murari SK. A non-cytotoxic pseudo peptide KL1 with potent antioxidant activity. Asian Journal of Research in Chemistry. 2017;10(2):127-30. doi : 10.5958/0974-4150.2017.00020.7

 

 

 

 

 

Accepted on 14.12.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(11):5295-5301.