1. INTRODUCTION:

Arthritis is one of the foremost health troubles worldwide, leading causes of disability in western and developing countries. Therapies developed along the principles of western medicine are often limited in efficacy, carry the risk of adverse effects, and are often too costly, especially for the developing world^[1]. Therefore, considering treatment for arthritis with herbal and herbal products is alternative and safe therapy.

Aegle marmelos (AM) is a slow-growing, medium sized tree, which is commonly distributed in India especially in temple of Tamil Nadu. The Tamil Nadu people traditionally believe this plant is God’s plant (Lord Shiva in Hindu religion)^[2,3]. Different organic extracts of the leaves of AM have been reported to possess alkaloids, cardiac glycosides, terpenoids, saponins, tannins, flavonoids and steroids. AM fruit pulp reported for the availability of steroids, terpenoids, flavonoids, phenolic compounds, lignin, fat and oil, inulin, proteins, carbohydrates, alkaloids, cardiac glycosides and flavonoids^[4]. Many of the Ayurveda formulations for arthritic treatment contains the AM, but since there is no scientific evidence for the antiarthritic activity of AM. Therefore, current research is aimed to evaluate the antiarthritic activity of AM with different solvent extraction.

2. Materials and methods:

2.1. Plant Collection and Authentication:

The fresh unripe fruits of the plant AM were collected from Sri Mariamman Temple, Kalapatti, Coimbatore District, Tamil Nadu, and India, Month of January 2019, and authenticated by Botanical survey of India (BSI) southern circle, Coimbatore, Tamil Nadu. The authentication certificate number is No. BSI/SRC/ 5/23/2019/Tech/108.

2.2. Preparation of plant material:

The collected unripe fruits were cleaned, dried under sunshade in dark room, and powdered by using mechanical mixer. After size reduction fruit powder were sieved under sieve No. 40 and sieve No. 60, stored in airtight container at room temperature^[5,6].

2.3. Extraction of the plant material:

About 15 g of plant materials were extracted with 150 ml of different solvents (petroleum ether, chloroform, ethyl acetate, methanol, water) based on low polarity to high polarity by cold maceration method for 48 h. After extraction the extracts were separately concentrated by distillation and dried at room temperature until get viscous solid mass. The obtained crude extracts were weighed and stored at 4⁰C for the further analysis^[7,8]. The percentage yield was calculated by using following formula

Weight of extract obtained (g)

Percentage Yield (%w/w) = –––––––––––––––––––––––––––––×100

Weight of plant material used

2.4. Preliminary phytochemical analysis:

The obtained extracts were named as PEEAM for petroleum ether extract, CEAM for chloroform extract, EAEAM for ethyacetate extract, MEAM for methanolic extract and AEAM aqueous extract. All the different extracts were subjected to phytochemical evaluation and identified the various plant constituents present in the test sample qualitatively^[9,10,11].

2.5. In vitro anti-oxidant studies:

2.5.1. DPPH free radical scavenging assay:

The radical scavenging capacity of the extracts on DPPH radical was evaluated. About 1ml of 0.135 mM DPPH in methanol was mixed with 1ml of different extract (0.02–0.1mg concentration in respective solvents). The mixture was incubated for a period of 30 min in the dark at room temperature. After the incubation period absorbance was measured at 517nm in spectrophotometer. Quercetin was used as standard.^[12,13] The radical scavenging activity was calculated by, DPPH radical scavenging activity (%) = [(AbsControl - AbsSample)]/(AbsControl)] x 100 where AbsControl is the absorbance of DPPH radical with methanol; AbsSample is the absorbance of DPPH radical and either the sample extract or standard.

2.5.2. ABTS radical scavenging assay:

To determine ABTS radical scavenging assay, the stock solutions included 7 mM ABTS solution and 2.4 mM potassium persulfate solution^[14]. Both the reagents were mixed in equal quantities for preparing the working solution. And allow it to react for 12 h at room temperature in the dark. Further the mixture was diluted by mixing one ml ABTS solution with 60ml of methanol so that the absorbance becomes 0.706±0.001 units at 734 nm using the spectrophotometer. Fresh ABTS solution was prepared for each assay. Plant extracts (1000µl) were allowed to react with (1000µl) of the ABTS solution for 7 min and the absorbance was read at 734nm. The ABTS scavenging capacity of the extract was compared with that of quercetin and percentage inhibition was calculated using the formula [(Abs Control- Abs Sample)]/(Abs Control) x100 where, Abs Control is the absorbance of ABTS radical and methanol, AbsSample is the absorbance of ABTS radical and sample extract or standard. Percentage inhibition (I %) = (Abs control- Abs sample/Abs control) X 100. Different sample concentrations were used in order to obtain calibration curves and to calculate the IC50 values^[15].

2.6. In vitro anti-arthritic activity:

2.6.1. Bovine serum denaturation method:

About 0.05ml of various concentrations (50, 100, 200µg/ml) of different extracts and standard drug diclofenac sodium (50, 100, 200µg/ml) were taken respectively and 0.45ml (0.5%w/v BSA) mixed. The samples were incubated at 37⁰C for 20 minutes and the temperature was increased to keep the samples at 57⁰C for 3 minutes. Then 2.5ml of phosphate buffer was added to the above solution. The absorbance was measured using UV –visible spectrophotometer at 255nm^[16]. The control represents 100% protein denaturation. The results were compared with diclofenac sodium^[17]. The percentage inhibition of protein denaturation can be calculated as

Percentage inhibition=100-[optical density of test solution -optical density of control] X 100.

2.6.2. Egg albumin denaturation method:

About 0.2ml of egg albumin (from fresh hen’s egg), 2.8 ml of phosphate buffered saline (pH 6.4) and 2ml of different concentrations (50, 100, 200µg/ml) of different extracts were mixed to form 5ml of reaction mixture. A similar volume of double distilled water served as a control^[18]. Next the mixtures were incubated at 37±2⁰C in a BOD incubator for 15 min and then heated at 70⁰C for five min, after cooling their absorbance was measured at 600nm by using the vehicle as a blank. Diclofenac sodium in the concentrations of 50, 100, 200 µg/ml was used as the reference drug and treated similarly for the determination of absorbance. The % inhibition of protein denaturation was calculated by using the following formula. % inhibition = 100 [A1-A2/A1]. Where, A1=absorbance of control, A2 = absorbance of the test sample. The extract concentration for 50% inhibition (IC₅₀) was determined by the dose response curve^[19].

3. Results:

3.1. Extractive yield:

The percentage yield of different extracts of AM was shown in Table 1. From the results Aqueous extract shows higher yield of extract 15.08% w/w when compared with other extracts and petroleum ether extract has lower extractive yield which indicates the petroleum ether has poor extractive capacity of AM.

Table 1: Percentage yield of different extracts of AM

Extract	Amount of Plant material used (g)	Amount of extract obtained (g)	Percentage yield (% w/w)
PEEAM	15	0.723	4.82
CEAM	15	0.971	6.47
EAEAM	15	0..889	5.92
MEAM	15	1.902	12.68
AEAM	15	2.262	15.08

3.2. Phytochemical analysis:

Preliminary phytochemical evaluation of different extracts of AM was shown in Table 2. The results revealed that MEAM, AEAM, EAAM and CEAM have alkaloids, Flavonoids, carbohydrates, glycosides, proteins and amino acids. Saponins present in CEAM, EAEAM and MEAM. PEEAM have only of steroids and triterpenoids and absence of tannins in all the extracts.

Table 2: Preliminary phytochemical evaluation of different extracts of AM

Phyto constituents	PEEAM	CEAM	EAEAM	MEAM	AEAM
Alkaloids	Absent	Present	Present	Present	Present
Flavonoids	Absent	Present	Present	Present	Present
Tannins	Absent	Absent	Absent	Absent	Absent
Steroids	Present	Absent	Absent	Absent	Absent
Triterpenoids	Present	Absent	Absent	Present	Absent
Carbohydrates	Absent	Present	Present	Present	Present
Saponins	Absent	Present	Present	Present	Absent
Glycosides	Absent	Present	Present	Present	Present
Proteins and amino acids	Absent	Present	Present	Present	Present

3.3. In vitro antioxidant assay:

The percentage inhibition of DPPH radical scavenging assay and ABTS radical scavenging assay of different extracts were shown in Figure 1 and Figure 2 respectively. The percentage inhibition of standard quercetin and plant extracts were increased with the increasing of concentration. The petroleum ether extract shows least activity when it was compared with all other extracts and aqueous extract has higher activity which is produced the similar activity with quercetin.

Figure 1: DPPH scavenging activities of different plant extracts and standard Quercetin

Figure 2: ABTS scavenging activities of different plant extracts and standard Quercetin

IC ₅₀ of different extract was calculated using the calibration curve of respective extracts which was mentioned in Table 3. The petroleum ether extract has the higher IC ₅₀ value compared with other extracts, and aqueous extract shows lower IC ₅₀ values and which has similar activity of quercetin.

Table 3: DPPH and ABTS radical scavenging IC ₅₀ Value of different extracts and standard

Plant extract/ Standard	DPPH IC ₅₀(µg/ml)	ABTS IC ₅₀(µg/ml)
Quercetin	23.69	13.512
PEEAM	151.90	71.92
CEAM	39.47	60.44
EAEAM	109.77	55.53
MEAM	36.69	54.46
AEAM	30.74	27.29

3.4. Antiarthritic activity:

Antiarthritic activity was evaluated by BSA protein denaturation method and egg albumin protein denaturation method. Percentage inhibition of BSA protein denaturation method and egg albumin protein denaturation was shown in Figure 3 and Figure 4.

IC₅₀ value of different extract of BSA denaturation method and Egg albumin denaturation method was shown in Table 3. The petroleum ether extract has the higher IC₅₀ value compared with other extracts, and aqueous extract shows lower IC₅₀ values and which has similar activity of Diclofenac sodium.

Table 4: BSA Protein denaturation inhibition and Egg albumin denaturation IC ₅₀ value of different extracts and standard

Extract/ Standard	BSA denaturation IC ₅₀(µg/ml)	Egg albumin denaturation IC₅₀ (µg/ml)
Diclofenac sodium	15.716	12.86
PEEAM	48.26	46.09
CEAM	43.29	38.09
EAEAM	31.36	34.86
MEAM	42.23	37.99
AEAM	28.104	19.85

4. Discussion:

Arthritis is one of the leading health problems worldwide. Treatments established along the principles of western medicine are often limited in effectiveness, Carry the risk of adverse effects, and are often too expensive, especially for the emerging world. Therefore, treating arthritis with plant and plant derived compounds are alternative treatment with safety and less cost. The medicinal plant has secondary metabolites (phytochemicals) for their medicinal value. For example, triterpenoids, flavonoids and related polyphenols have antiarthritic properties^[20].

The results of present study, phytochemical analysis of different extracts of AM revealed the presence of various bioactive phytochemical compounds was found in aqueous, methanolic, ethylacetate, chloroform and petroleum ether extracts. In vitro anti-arthritic activity of PEEAM, CEAM, EAEAM, MEAM and AEAM were screened against protein denaturation against bovine serum albumin and egg albumin. The unriped fruit extract of AM showed significantly higher anti-arthritic activity at increasing concentration. The possible mechanism of antioxidant and antiarthrtritic activity may due to the presence of active principles such as flavonoids, alkaloids and triterpenoids and polyphenols present in AM. Hence, AM can be used as an anti-arthritic agent^[21].

5. Conclusion:

Results of the current research work revealed that unripe fruit extract of AM has antiarthritic activity and aqueous extract of AM has potent antiarthritic and antioxidant activity. This research work is useful to further isolation of active principles and study of molecular mechanism of aqueous extract of AM.

6. List of symbols and Abbreviations:

AM: Aegle marmelos (L.) Correa

BSI: Botanical survey of India

PEEAM: Petroleum ether extract of Aegle marmelos

CEAM: Chloroform extract of Aegle marmelos

EAEAM: Ethyacetate extract of Aegle marmelos

MEAM: Methanolic extract of Aegle marmelos

AEAM: Aqueous extract of Aegle marmelos

DPPH: 2,2-diphenyl-1-picrylhydrazyl

ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid

BSA: Bovine serum Albumin

IC ₅₀: 50% inhibitory concentration

7. Acknowledgement:

The authors thank to Dr. A. Rajasekaran, Principal, KMCH College of Pharmacy who has provided excellent facilities to do research in this institution and our beloved Managing Trustee, Dr. Nalla G. Palaniswami and respected Trustee madam Dr. Thavamani D. Palaniswami, Kovai Medical Center Research and Educational trust, Coimbatore for all the facilities that were provided at the institution.

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Received on 26.08.2019 Modified on 01.10.2019

Research J. Pharm. and Tech 2020; 13(6): 2759-2763.

DOI: 10.5958/0974-360X.2020.00490.4