Natural Capsaicin, Honey and Its Combinatory effect in Complete Freund's adjuvant Induced Arthritis in Rats

Ramandeep Kaur¹, Pankaj Kumar¹, Amit Lather²*, Sachin Kumar³, Ramchander Khatri⁴,

Dharmbir Singh⁵, Sandeep Kumar¹, Tanuj Hooda¹*

¹MM College of Pharmacy, Maharishi Markandeshwar (Deemed to be University),

Mullana - Ambala, Haryana, India -133207.

²Geeta Institute of Pharmacy, Geeta University, Naultha, Panipat, Haryana, India -132145.

³Bharat Institute of Pharmacy, Rurki, Kurukshetra, Haryana, India – 136132.

⁴Delhi Pharmaceutical Sciences and Research University, New Delhi, Delhi 110017.

⁵Faculty of Pharmacy, RP Educational Trust, Badshara, Karnal, Haryana. India -132114.

*Corresponding Author E-mail: tanujhooda2010@gmail.com, dramitlather@gmail.com

ABSTRACT:

Background: Rheumatoid arthritis is disease of concern in today’s population, due to their lifestyle, smoking, stress, obesity. As herbal drugs have lesser side effects, and chilli and honey are being used by global population from generations. But as chilli alone could cause burning sensation, very hard to swallow, and excessive use could cause gastric ulceration, but in combination with honey, the burning sensation caused by chilli and honey have healing properties too. Methods: Anti-inflammatory activity of capsaicin was evaluated in Arthritis induced rat models. Result: The reduction in inflammation, Plethysmometer was used to measure inflammation every week, and it showed decline in inflammation, with declining in body weight too. The group of animals where administered dose was hydro-alcoholic extract at 60 mg/ kg with honey after 28 days show no symptoms of arthritis, whereas other groups showed low to mild arthritis but their level of inflammation was very low as compared to positive disease group. Conclusion: At last, it has been concluded that Capsaicin extract if given in combination with honey shows better anti-inflammatory results. But in combination with honey there is better reduction in inflammation due to cytoprotective and wound healing properties.

KEYWORDS: Rheumatoid arthritis, Capsaicin, Honey, Histopathological, Plethysmometer, FCA, Chilli.

INTRODUCTION: 

Rheumatoid arthritis (RA) is a chronic, systemic, inflammatory autoimmune disease with progressive degradation of large, small joints as well as other organs, generally being observed between the ages of thirty to fifty years¹. It is the most common inflammatory disease which affected 25 men and 54 women per 100,000 populations and remained the main cause for hospitalizations of 250,000 patients with over 9 million medical practitioner visits within the U.S. annually². The main subtypes of RA consistent with the presence or absence of anti-citrullinated macromolecule antibodies (ACPAs) also called seropositive and seronegative^2,3.

The ACPA-positive set of RA features an additional aggressive clinical composition compared to ACPA-negative charge of RA⁵. It is reportable that ACPA-negative RA has totally different genetic association patterns. In terms of treatment, there is less effective treatment response by immune suppressant drug (MTX) or rituximab in ACPA-negative set⁶.

Chilli belongs to asteria dicot family. The main active ingredient of Chilli is Capsaicin (C₁₈H₂₇NO₃), this is the main reason behind the pungency of chilli. The main applications of capsaicin are antioxidant⁷, anticancer^8-10, anti-inflammatory, and analgesic^11-13. The main phytochemicals of chilli are proteins, tryptophan, threonine, isoleucine, valine, arginine, histidine, glutamic acid, glycine, proline, Serine, vitamins, minerals¹⁴. Chilli having zero caloric value and includes carbohydrates, fats, andproteins¹⁵. It also contain fats and fatty acids, saturated fat, mono-unsaturated fat, poly-unsaturated fat, omega- 3 fatty acids, and omega- 6 fatty acids, etc. As fatty acids are obtained from fats and their main role is to provide energy¹⁵. The main carbohydrates present are dietary fibre and sugars¹⁶. The most common types of proteins and amino acids which are present in chilli are proteins, tryptophan, threonine, isoleucine, leucine, lysine, methionine, cystine, phenylalanine, tyrosine, valine, arginine, histidine, alanine, aspartic acid, glutamic acid, glycine, proline, and serine¹⁷. It contains vitamin A, retinol. Retinol activity equivalent, alpha-carotene, beta-carotene, beta-cryptoxanthin, lycopene, lutein, zeaxanthin, vitamin C, vitamin E (a-tocopherol), vitamin K, thiamine, riboflavin, and niacin¹⁸. It contains various minerals like calcium, iron, magnesium, phosphorus, potassium, sodium, zinc, copper, manganese, selenium, and fluoride¹⁹.

Honey is a natural product obtained from bees having anti-inflammatory and wound healing medicine²⁰. Honey contains approximately 200 compounds, such as sugar, protein, enzymes, minerals, vitamins, amino acids, polyphenols, mono-saccharides etc. Invertase, glucose oxidase, and diastase are the main enzymes in honey²¹. The ingredients of honey are responsible for, antioxidant^22-24, antiproliferative^25-26, anti-inflammatory^27-29 and anti-metastatic effects³⁰. Previous studies have detected some 600 volatile compositions in honey that contribute to its potential medication effects. The volatile compounds of honey unit are typically low however it contains aldehydes, alcohols, hydrocarbons, ketones, acid esters, aromatic hydrocarbon and its derivatives, chemical compound and its derivatives, nor-isoprenoids, sulphur, furan, and cyclic compounds. Flavonoids and polyphenols, which act as antioxidants is also present³¹.

MATERIAL AND METHODS:

First of all, plant of chilli was selected and authentication. The animal experiment protocol was approved by the IAEC, MM(DU), Mullana, Ambala with approval number MMCP-IAEC-24/2020 and all the experimental procedures were carried out under the guidelines of the CCSEA. All the chemicals used in the study were of analytical grade.

Biological Name - Capsicum annuum L.; Family– Solanaceae; Common names - Chilli pepper, hot pepper, bell pepper, etc. (Figure 1) Chemical Structure of capsaicin shown in Figure 2.

Figure 1: Capsaicin Plant

Figure 2: Chemical Structure of capsaicin

Procedure:

Green chillies were taken from the Capsicum annuum L., which was authenticated. The chillis were collected, crushed coarsely and divided into two equal halves. Then each halve is placed into a different Soxhlet apparatus one containing chloroform extract, where, solvent is heated at 30 – 40℃ to reflux, solvent vapour travels up through distillation arm, and floods into the chamber housing the thimble, the condenser cools the vapour and drips back into the chamber containing thimble. The chamber containing the solid material slowly fills with warm solvent and desired compounds would get dissolved into the solvent. When the Soxhlet chamber is almost full, the chamber gets automatically emptied by a siphon tube, by the process of siphoning. This cycle gets repeated many times until the colour of solution in siphon tube gets transparent. After extraction solvent is removed, the non-soluble portion of the extracted solid remains in the thimble is usually discarded. Similarly, Hydro-Alcoholic solvent was used for another half powder, the hydro-Alcoholic (80% water + 20% Ethanol), at Temperature 30 - 40℃ is used in place of chloroform to get the extract.

After extract was obtained, phytochemical analysis was performed on these extracts- including test for alkaloids, saponins, glycosides, flavonoids, tannins, synthetic resin compounds, carbohydrates, reducing sugars, fixed oils and fats. These tests were performed on both extracts by using 2 methods in each test^32-33, so that test results would be more accurate. 

Induction of Arthritis:

Arthritis in Wistar rat was induced by FCA, i.e. Freund’s complete adjuvant. It is a commonly used method in Rats. Dose of 0.1 ml was administered to the Wistar rat by intradermal injection into the right hind metatarsal foot paw. 9 group of animals with 6 animals in each group were taken. FCA was administered into 8 groups whereas 1 group was normal.

RESULTS AND DISCUSSION:

Phytochemical analysis- These tests were performed by using 2 different tests, like for checking the presence of alkaloids, in place of 1 test, 2 tests were performed Dragendorff Test, and Hager's Test, so that less chance of error would be there and 2 samples i.e., chloroform solvent extract and hydro-alcoholic solvent extract were analyzed differently and analysis of Phytoconstituents were reported in Table1.

In vivo study:

The wistar rats were divided as 6 animals in a group with the standard deviation. As the study was of 28 days, the weight was measured after every 7 days of dosing, starting from the day 0, i.e., the day the arthritis as induced by FCA, till the last day of the treatment shown in Table 2 and Graphical representation shown in Figure 3.

Table 1: Phytochemical tests for Chloroform sample and Hydroalcoholic sample of Capsaicin.

S. No	Phytoconstituents	Tests performed	Chloroform extract	Hydro-alcoholic extract
	Alkaloids	Dragendorff Test	+	-
		Hager's Test	+	-
	Saponins	Froth Test	+	+
		Foam Test	+	+
	Fixed oils and Fats	Spot Test	+	-
		Saponification Test	+	-
	Carbohydrates	Molisch Test	+	-
	Reducing sugar	Benedict's Test	-	+
		Fehling Test	-	+
	Flavonoids	Ferric chloride Test	+	+
		Alkaline Reagent Test	+	+
	Glycosides	Killer Killani Test	-	+
		Bromine Water Test	-	+
	Tannins	Gelatin Test	+	+
		Test with Phenazone	+	+
	Phenolics	Ferric chloride Test	-	+
		Lead acetate Test	-	+

“+” Sign indices the presence of phytoconstituent and “-” Sign indicates the absence of phytoconstituent

Table 2: Weight of Wistar Rat with time in different groups.

Date	Day 0	Day 7	Day 14	Day 21	Day 28
Normal group	236.4 ± 5.3	242.6 ± 8.2	246.4 ± 10.7	253.1 ± 9.6	257.2 ± 11.2
Positive disease group	263.5 ± 5.2	260.6 ± 7.5	258.2 ± 8.9	261.9 ± 9.6	264.4 ± 10.3
Standard Methotrexate 7.5 mg/ kg	253.5 ± 5.8	249.9 ± 7.6	247.7 ± 8.4	250.2 ± 9.3	256.4 ± 10.7
Chloroform extract 30 mg/ kg + 0.1 ml Honey (50%)	272.3 ± 4.3	269.7 ± 5.7	265.1 ± 8.4	262.9 ± 7.8	258.6 ± 6.2
Chloroform extract 60 mg/ kg + 0.1 ml Honey (50%)	249.4 ± 7.4	244.6 ± 8.9	239.7 ± 9.5	234.7 ± 9.2	228.2 ± 11.8
Chloroform extract 120 mg/ kg + 0.1 ml Honey (50%)	278.7 ± 7.7	270.2 ± 6.3	261.6 ± 8.5	252.8 ± 8.7	243.4 ± 9.2
Hydroalcoholic extract (20: 80) 30 mg/ kg + 0.1 ml Honey (50%)	272.4 ± 7.4	269.3 ± 8.3	264.7 ± 9.7	261.2 ± 9.1	258.5 ± 11.3
Hydroalcoholic extract (20:80) 60 mg/ kg + 0.1 ml Honey (50%)	266.7 ± 7.4	261.3 ± 8.2	256.2 ± 9.3	250.7 ± 11.5	246.9 ± 12.6
Hydroalcoholic extract (20:80) 120 mg/ kg + 0.1 ml Honey (50%)	238.4 ± 6.2	233.6 ± 7.3	227.2 ± 8.5	221.3 ± 8.6	214.7 ± 9.7

Figure 3: Graphical representation of groups (1-6) of animal weight according to time intervals and Graphical representation of groups (1-3 and 7-9) of animal weight with time intervals.

In the graph 1 denotes Normal group, 2 denotes Positive disease group, 3 denotes group treated with Standard Methotrexate 7.5mg/kg, 4 denotes group treated with Chloroform extract 30mg/kg + 0.1ml Honey (50%), 5 denotes group treated with Chloroform extract 60 mg/ kg + 0.1 ml Honey (50%), 6 denotes group treated with Chloroform extract 120mg/ kg + 0.1ml Honey (50%), The graph 1,2,3 is compared with 4, 5, and 6.

And 7 denotes group treated with Hydroalcoholic extract (20:80) 30mg/kg + 0.1ml Honey (50%), 8 denotes group treated with Hydroalcoholic extract (20:80) 60mg/kg + 0.1ml Honey (50%), 9 denotes group treated with Hydroalcoholic extract (20:80) 120mg/kg+0.1ml Honey (50%). The graph 1,2,3 is compared with 7, 8 and 9, and it is plotted between weights in grams vs Time in days.

The inflammation was measured in ml, where it was the average of the group of 6 animals of a group, As the study was 28 days long, inflammation was measured after every 7 days with the help of digital Plethysmometer, starting from day 0, i.e., when arthritis was induced by FCA, till last day of dosing that is 28^th shown in Table 3 and Graphical representation shown in Figure 4.

In the graph above 1 denotes Normal group, 2 denotes Positive disease group, 3 denotes group treated with Standard Methotrexate 7.5mg/kg, 4 denotes group treated with Chloroform extract 30mg/kg + 0.1ml Honey (50%), 5 denotes group treated with Chloroform extract 60mg/kg + 0.1ml Honey (50%), 6 denotes group treated with Chloroform extract 120mg/kg + 0.1ml Honey (50%), The graph 1, 2, 3 is compared with 4, 5, and 6.

Table 3: Inflammation of Wistar Rat by using Plethysmometer.

Date	Day 0	Day 7	Day 14	Day 21	Day 28
Normal group	1.3 ± 0.05	1.32 ± 0.09	1.36 ± 0.14	1.36 ± 0.11	1.38 ± 0.07
Positive group	1.8 ± 0.08	2.86 ± 0.11	2.8 ± 0.07	2.72 ± 0.14	2.6 ± 0.09
Standard Methotrexate 7.5mg/ kg	1.58 ± 0.06	2.24 ± 0.09	1.82 ± 0.13	1.66 ± 0.10	1.6 ± 0.16
Chloroform extract 30mg/kg + 0.1ml Honey (50%)	1.95 ± 0.07	2.84 ± 0.16	2.67 ± 0.21	2.45 ± 0.11	2.38 ± 0.14
Chloroform extract 60mg/kg + 0.1ml Honey (50%)	1.44 ± 0.07	2.28 ± 0.14	2.02 ± 0.18	1.8 ± 0.11	1.46 ± 0.07
Chloroform extract 120mg/ kg + 0.1ml Honey (50%)	2.04 ± 0.11	2.95 ± 0.07	2.56 ± 0.18	2.24 ± 0.16	2.06 ± 0.18
Hydroalcoholic extract (20: 80) 30mg/kg + 0.1ml Honey (50%)	1.94 ± 0.09	2.8 ± 0.15	2.54 ± 0.19	2.32 ± 0.14	2.08 ± 0.11
Hydroalcoholic extract (20:80) 60mg/kg + 0.1ml Honey (50%)	1.78 ± 0.14	2.69 ± 0.09	2.36 ± 0.22	1.98 ± 0.14	1.8 ± 0.16
Hydroalcoholic extract (20:80) 120mg/kg + 0.1ml Honey (50%)	1.31 ± 0.06	1.92 ± 0.15	1.7 ± 0.18	1.52 ± 0.14	1.32 ± 0.21

Figure 4: Graphical representation of inflammation in animals (1-6) and Graphical representation of inflammation in animals (1-3 and 7-9).

And 7 denotes group treated with Hydroalcoholic extract (20: 80) 30mg/kg + 0.1ml Honey (50%), 8 denotes group treated with Hydroalcoholic extract (20:80) 60 mg/ kg + 0.1ml Honey (50%), 9 denotes group treated with Hydroalcoholic extract (20:80) 120mg/kg + 0.1ml Honey (50%). The graph 1, 2, 3 is compared with 7,8, and 9, and it is plotted between inflammation vs Time in days.

Results of histopathology of the tissue indicated normal tissue with no sign of inflammation, arthritis mean- huge inflammation in the tissues, whereas mild arthritis mean inflammation is mild or non-serious shown in Table 4 (Figure 5).

Figure 5: Histopathology of Tissue.

Table 4: Histo-Pathology of the tissue of Right leg of Rat (Impression)

S. No	Group of animals	Leg Impression
	Normal group	Descriptive
	Positive disease group	Arthritis
	Standard Methotrexate 7.5mg/ kg	Descriptive
	Chloroform extract 30mg/ kg + 0.1ml Honey (50%)	Mild arthritis
	Chloroform extract 60mg/ kg + 0.1ml Honey (50%)	Mild arthritis
	Chloroform extract 120mg/ kg + 0.1ml Honey (50%)	Mild arthritis
	Hydroalcoholic extract (20:80) 30mg/ kg + 0.1ml Honey (50%)	Mild arthritis
	Hydroalcoholic extract (20:80) 60mg/ kg + 0.1ml Honey (50%)	Descriptive
	Hydroalcoholic extract (20:80) 120mg/ kg + 0.1ml Honey (50%)	Mild arthritis

Here, current research explores the potential of two types of capsaicin extracts i.e. chloroform and Hydro-alcoholic at different doses in the treatment of arthritis. Results indicated the reduction in inflammation in rat model, Plethysmometer was used to measure inflammation every week, and it showed decline in inflammation, with declining in body weight too. After completion of 28 days study, the tissue from the right hind paw of the animals were also collected for histo- pathological analysis. The group of animals where administered dose was hydro-alcoholic extract at 60mg/ kg with honey after 28 days show no symptoms of arthritis, whereas other groups showed low to mild arthritis but their level of inflammation was very low as compared to positive disease group, where no treatment was given and also to the group of animals where standard marketed formulation (Methotrexate) was administered. This indicated that drug show positive response against arthritis, it lowered the inflammation to all the groups.

CONCLUSION:

It has been concluded from that capsaicin extract in combination with honey shows better anti-inflammatory results. As it was mentioned in literature about their effect on arthritis, there is market formulation of Capsaicin available in market. But orally capsaicin may cause gastric irritations and ulcers too, but when capsaicin could be used in combination with Honey due to better tolerability, reduced inflammation, cytoprotective and have wound healing properties too. So, the combined effect of honey with Capsaicin showed better results in rheumatoid arthritis. As plants have very important role in the treatment of arthritis^34-40, although further research required to clarify the potential of capsaicin and honey in the treatment of rheumatoid arthritis.

ACKNOWLEDGEMENT:

I owe special thanks to Prof (Dr.) Randhir Singh Dahiya of Maharishi Markandeshwar University,Deparament of pharmacy (Pharmacology), for giving me their genuine support and valuable guidance throughout the study.

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Received on 06.04.2024      Revised on 02.08.2024

Accepted on 08.10.2024   Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):5707-5712.

DOI: 10.52711/0974-360X.2024.00869

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