Sugar Palm Fruit (Arenga pinnata) Diminish Pain and Inflammatory Symptoms of Osteoarthritis in male Wistar Rat (Rattus norvegicus)

 

UmiKalsum¹, Husnul Khotimah¹, Wibi Riawan², Hasnia Azizah Trisnaini³, Elok Zubaidah³

¹Departement of Pharmacology, Medical School, Universitas Brawijaya, Indonesia

²Departement of Biochemistry and Molecular Biology, Medical School, Universitas Brawijaya, Indonesia

³Agricultural Technology Faculty,Universitas Brawijaya, Indonesia

 

ABSTRACT:

Sugar palm fruit is the seed of Aren fruit (Arenga pinnata) that contain galactomannan polysaccharides. Galactomannan is the polysaccharide that has analgesic activity. The purpose of this research was to determine the effect of analgesic and anti-inflammation of sugar palm fruit juice to the inflammation induced by 0.6% acetic acid and Complete Freund's Adjuvant (CFA) to male Wistar rats (Rattus norvegicus) respectively. Sugar palm fruit juice made by blending the sugar palm fruit and water which ratio 1:2. The male rats were divided into 6 groups: control, induced with acetic acid 0.6% (analgesic) or CFA (inflammation), induced with acetic acid 0.6% (analgesic) or CFA (inflammation) and sodium diclofenac 10 mg/200 g BW of rats, and last three groups were induced by acetic acid or CFA and administered by sugar palm fruit extract dose 1, 2 and 3 mg/200 g BW.  We used Fourier Transform Infrared (FITR) analysis to identify molecular structure of galactomannan in sugar palm fruit juice. The results showed that sugar palm fruit juice reduced pain and edema in rats. The highest efficacy of analgesic and anti-inflammatory response was at 3 ml/200 g BW sugar palm fruit juice which potension of analgesic was 69.7% and anti-inflammatory response to decrease edema volume was 1,608 ml. FITR peaks showed that galactomannan had main chain β-(1-4)-D_mano-pyranose and bind to D-galacto pyranose in α-1-6 chain. It could be concluded that sugar palm fruit juice potential to developed as analgesic and anti-inflammatory agent.

 

 

 

INTRODUCTION:

Sugar palm fruit (Arenga pinnata) is widely used by Indonesian people as a drink. The availability of sugar palm fruit (SPF) abundant because of its spread almost in all area of ​​Indonesia [1]. The chemical composition of sugar palm fruit is dominated by carbohydrates (52.9%). Carbohydrates in SPF have a polysaccharide component of galactomannan that serves as a pain reliever (analgesic) and anti-inflammatory [2]. Galactomannan consists of β- (1-4)- mannose backbone having single α- (1-6)- galactose side chain.

 

The galactomannans are unique, compared to the more common polysaccharides by differing in spatial dispositions of their OH groups. Mannose and galactose, each have a pair of OH groups on the same side (cis position) of the pyran ring [3]. Osteoarthritis is a chronic degenerative disease that occurs in joints characterized by a mixture of degradative and reparative processes in the articular cartilage, subchondral bone associated with marginal osteophyte formation, and low grade inflammation that cause pain and inflammation [4], [5]. Inflammation is a most troublesome disease, and has a substantial effect in terms of cost, disability, and low productivity. It affects 1% of world population and is associated with significant morbidity and mortality [6]. Inflammation is a protective attempt by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue. The nature of the inflammatory response following tissue injury depends upon both the time elapsed since injury as well as the extent of tissue damage [7]. Pain and inflammation are symptoms of illness due to tissue damage or tissue metabolism disorders followed by the release and formation of the mediator of inflammation, such as histamine, serotonin, and bradykinin [8]. Prevalence of OA increases with age, so that about 11% of all women over the age of 60 yr have symptoms due to knee OA [9]. Electromagnetic fields (EMFs) have shown positive therapeutic effects in this disorder. Several studies have investigated the effectiveness of different EMFs for the treatment of OA with controversial [10]. Standard treatment of osteoarthritis so far was with the provision of DMARD's (Disease Modifying Anti Rheumatic Drugs), namely drugs that are strong anti-inflammatory and often combined with NSAID's (Non-Steroid Anti-Inflammatory Drugs) that serves to support its performance. However, in the long term usage of NSAID’s, its have other toxic side effects. NSAIDs can increase the risk of heart attacks, strokes, kidney and toxic for blood that lead to death and internal stomach bleeding during treatment [11].  Reddy, et al [12] proved that Curcuma longa have anti-inflammatory action on IL-1beta-induce osteoarthristis in chondrocytes through the suppression of NFkB pathway. Sugar palm fruit proposed has potential galactomannan property as a pain reliever and anti-inflammatory in patients with osteoarthritis. Experiments were performed on pain-induced rats using acetic acid and chronic inflammation using Complete Freund's Adjuvant (CFA) and then treated with SPF juice to determine the effect on analgesics and anti-inflammatory. In addition, the treatment of SPF juice can be applied for daily consumption in humans. The purpose of this study was to determine the effect of analgesic and anti-inflammatory on the stretching responses caused by acetic acid and Complete Freund's Adjuvant (CFA) in male Wistar rats (Rattus norvegicus).

 

MATERIAL AND METHODS:

Chemicals and reagents:

The main ingredients used in this study is SPF obtained from the traditional market in Pasar Besar Malang East Java, Indonesia. The chemicals used were aquades (Otsuka), Ethanol 96% and Petroleum Eter (E-merck), 0.6% acetic acid (E-merck). In vivo test material used was male wistar rats (Rattus norvegicus) from laboratoty of Pharmacology Medical Faculty UniversitasBrawijaya, 10 weeks with susceptible weight 160-200 gram. The rats were divided into 6 groups :

P0: Normal control (given aquadest)

P1: Negative control was 0.6% acetic acid injected rats without any treatment;

P2: Positive controls was 0.6% acetic acid injected rats and given sodium diclofenac 10 mg / 200 gr BW;

P3: Rats injected by acetic acid 0.6% and given SPF juice dose 1 ml / 200 gr BW

P4: Rats injected by acetic acid 0.6% and given SPF juice dose 2 ml / 200 gr BW

P5: Rats injected by acetic acid 0.6% and given SPF juice dose 3 ml / 200 gr BW

 

Instrumentation:

The proposed work was carried out on a Shimadzu FITR 8400S, centrifuge (Heraeus 16r), valcon, sonde needle, cabinet dryer, analytical scale (Denver Instrument M-310), a rotary evaporator (Fisher Scientific).

 

The Analgesic Test of the SPF Juice Against the Acetic Acid Induction:

All protocols in this reaesrch have been approve by Ethical committe of Faculty of Medicine UniversitasBrawijaya. Qualitative analysis base on AOAC [13]. In the analgesic test, pain induction with 0.6% acetic acid 0.1 ml of intraperitoneal was then treated per oral of SPF juice in accordance with the dose. The given SPF juice (1,2 and 3 ml/200 BW) to adjust the volume of the rat's hull (<5 ml). Analgesic potency was calculated by determining the amount of stretching per five minutes for 70 minutes. The data were the cumulative stretching response of rats in each treatment group. Then calculated its analgesic potention using the formula by Turner [14].

 

The Anti-inflammatory Test of  SPF Juice Against CFA-induced Rat:

For the anti-inflammatory test, rats were induced by 0.1 ml Complete Freund's Adjuvant (CFA) intraplantar left foot which would induce chronic arthritis and inflammation. Tests of anti-inflammatory activity by looking at the activity of decreasing the volume of rat foot edema. Briefly, the procedure of anti-inflammatory measurement were : foot volume of the rat was measure by inserting the rat's legs on a water-filled syringe tube then viewing the volume of water, recorded as the initial volume. Observations were done every two days to determine the maximum volume of rat feet swelling. Administration of SPF juice dose of 1, 2 and 3ml / 200gBW, and 10 mg / 200gBW sodium diclofenac sodium were given when the volume of rat's legs gained twice than initial volume ie on the 8th day. Measurement of animal leg edema volume was attempted every 2 days for 1 month (30 days) because CFA induced chronic inflammation in the test rats. Each treatment group of test rats was calculated inflammation percentage with formula by Turner [14].

 

RESULTS AND DISCUSSION:

Chemical Compound Analysis:

The analysis of chemical compound of SPF juice was the proximate analysis which includes water content, ash, fat, protein, and carbohydrate. The results of the analysis of the chemical components of SPF juice can be seen in Table 1.

 

 Table 1.Proximate Analysis

Parameters	% Wet basis Sugar palm fruit
	Analysis	References[8]
Water (%) Ash (%) Protein (%) Fat (%) Fiber (%) Carbohydrate (%)	94,95 0,08 0,10 0,04 - 4,83	93,75 1,00 0,69 0 0,95 3,39

 

Carbohydrate analysis resulted in about 4.83% while literature [15] showed 3.39% which mean our SPF 1.44% higher than other. The main component of the SPF carbohydrate is galactomannan consisting of mannose's main chains and galactose branching chains. Galactomannan is a hemicellulose class polysaccharide which is the second most abundant plant biopolymer after cellulose. Galactomannan consists of a major chain of mannose with β 1-4 glycoside bonds and galactose branch chains of α 1-6 glycoside bonds. The number of galactose branches in each type of galactomannan varies by plant species [16]. Galactomannan extraction results are then calculated quantitatively to determine the yield of SPF. The result of galactomannan extraction of crude extract of SPF showed 2.57%, it means that 97.43% were other components besides galactomannan such as water, protein, fat and ash. The weight of galactomannan from 30 grams SPF extract was equal to 0.7705 grams.

 

FITR Analysis:

Fourier Transform Infrared (FT-IR) analysis is a qualitative analysis to identify the chemical structure of a molecule. FT-IR has a working principle of calculating the infrared light spectrum that is absorbed by chemical bonds as energy for vibration. The amount of absorbed infrared light spectrum is specific to each type of chemical bond, so that by knowing the FT-IR spectral profile of the material tested we can know what chemical bonds are in it, from the chemical bond type data present in the material we can identifying the components contained in the material.

 

The analyzed samples used rough extract of sugar palm fruit. Galactomannan analysis of SPF was performed by Shimadzu FT-IR spectrophotometer in wave length 400-4000 cm-1. The peaks generated from FT-IR vibrations can be seen in Figure 1

 

 

Figure 1 FT-IR spectra of Galactomannan from Sugar palm fruit extract

 

The extracts of galactomannan were analyzed qualitatively on chemical structures by FT-IR and the spectra can be seen in Fig. 1. Its showed that at 3402 cm-1 peak consist of O-H bond which is the polysaccharide composite macromolecule. The presence of a peak at frequencies 1084 cm-1 and 1026 cm-1 indicates the existed of a C-O bond in the carbonyl group at C-OH or at C-O-C. That was indicating a carbonyl group characteristic of the pyranose ring from the extract obtained. In addition, the finger print extract shows the frequency peak of 871 cm-1 and 812 cm-1 indicating the presence of α-galacto-pyranose binding units α and D-mano-pyranose units binding β, respectively. In general, galactomannan has a major chain of β- (1-4) -D-manopyranose bonds and attaches to D-galactopyranose at α-1-6 bonds.

 

Analgesic Test:

Analgesic testing was performed in vivo in rats (Rattus norvegicus). Chemical stimulation by injecting chemical compounds of 0.6% acetic acid intraperitoneally. Induction intraperitoneally performed to prevent the decomposition of acetic acid as it passes through the physiologic tissues of certain organs. The choice of acetic acid as a pain induction because of the injection triggers the activation of the cyclooxygenase pathway in arachidonic metabolism to produce prostaglandin (inflammatory and pain mediators). The positive response of pain was represented by stretching, defined as the body's stretching behavior, the pulling of the body to one side, the removal of the rear leg or abdominal restraint, so that the stomach touches the floor.

 

Acetic acid solution was administered shortly after the test compound was administered to the rats. The test compounds were SPF juice and sodium diclofenac given orally. This was done to make the test compound undergo an absorption phase to relieve pain. After induced of acetic acid, for a few minutes then the rats experienced pain indicated by a positive response. Observations made visually based on a predetermined standard form of stretching, the amount of rats stretched observed every 5 minutes for 70 minutes. The results of the observation can be shown in Figure 2.

 

 

Figure 2. Analgesic Power Test of Sugar palm fruit to rat stretching.

 

Table 2. Analgesic Test of Sugar palm fruit juice in Rats induced 0.6% acetic acid

Group	Mean  of stretching	Duncan	Notation	% Analgesic Potential	Standard Deviation
Negative Control	10,524	4,319	d	0	3,147
Normal Control	0,000	-	a	100	0,000
Sod-Diclofenac	6,310	3,819	c	40	1,204
Dose1 ml Sugar palm fruit juice/200 grBB	6,548	4,100	c	37,8	0,366
Dosis 2 ml Sugar palm fruit jiuce/200 grBB	5,643	3,428	bc	46,4	1,684
Dosis 3 ml Sugar palm fruit juice/200 grBB	3,191	2,801	b	69,7	0,743

 

 

Figure 2 showed a graph of analgesic test treatment against rat experimental animals. The presence of analgesic activity is indicated by the increase and decrease in the average amount of stretch produced. The smaller the average number of rats stretched, the greater the analgesic effect caused. Likewise, if the greater the average number of rat stretched, the smaller the analgesic effect caused by the treatment group.

 

Maximum stretching is shown in the negative control treatment at the 25th minute, Its because the negative control of rat induced 0.6% acetic acid without the use of drugs or SPF juice. Minimum stretching was shown in positive control treatment and SPF juice 3 ml/200 BW for 70 minutes. This was due to the sodium diclofenac as an anti-inflammatory standard drug. Diclofenac inhibits the thromboxane-prostanoid receptor, affect the arachidonic acid release and uptake, inhibit lipoxygenase enzymes, and activate the nitric oxide-cGMP anti-nociceptive pathway. Other novel mechanism of action may include the inhibition of substrate P, inhibition of peroxisome proliferator activated receptor gamma (PPARγ), blockage of acid-sensing ion channels, alteration of interleukin-6 production, and inhibition of N-methyl-D-aspartate (NMDA) receptor hyperalgesia [17]. SPF juice dose 3 ml/200 mg BW also had a lowered effect stretching in 70th minutes and equal to standard drug. The rats started to stretch in the 5th minute and tend to decrease at 40 to 70 minutes. were: is probably due to the ingredient of glucomannan in SPF juice. The results of the analgesic test can be shown in Table 2.

 

Based on the analysis of variance, we found that the administration of SPF juice could be decreased the analgesic test significantly. The highest analgesic response was performed by dose 3 ml/200 mg BW which equal to 69.7%. Negative control had minimal analgesic response equal to 37.8% which the rats have no treatment. The lower the average number of mouse stretches the higher the analgesic property. The administration of the crude extract of galactomannan Sugar palm fruit showed the analgesic response equal to 98,00% to the dose 200 mg/kg BW in rats [17].

Sugar palm fruit contains galactomannan which has the ability to inhibit the secretion of pain mediators, such as prostaglandin and/or bradykinin through arachidonic acid [18]. The principle of the mechanism is through the inhibition of the cyclooxygenase (COX)pathway. The secretion of the pain mediator is always followed by the activation of nociceptors in the joints [8], but the presence of galactomannan in SPF juice will inhibit the activation of the nociceptor.

 

Anti-inflammatory Test:

Anti-inflammatory tests were performed in vivo to the male Wistar rats (Rattus norvegicus). The test was performed by Complete Freund's Adjuvant (CFA) injection in rat's leg. The anti-inflammatory test was assessed through the decreasing volume of rat foot edema. The foot volume observed every 2 days for 30 days and daily oral treatment was performed on treatment groups. Oral treatment was done starting from day 8 because the osteoarthritis occurred. CFA contains an inactive mycobacterium that causes autoimmune disorders and systemic chronic inflammation. As well as trigger the formation of Reactive Oxygen Species (ROS) and free radicals that damage lipid membranes of cells, DNA and proteins, resulting in oxidative stress cells. It can suppress proteoglycan activity so that the production of polymorphonuclear cells increases and diffuses freely resulting in cartilage degradation and erosion that triggers edema.

 

The foot volume data then calculated to gain percent inflammation resulting from each treatment using the formula [14]. Percentage of inflammation is the percentage change in rat's foot volume by comparing the volume of rat's foot at certain time of the day compared to the rat's foot volume at 0 day before CFA-induced. Anti-inflammatory test results can be seen in Figure 3.It showed the volume of rat foot edema in ml (measurement using spuit syringe). The maximum edema volume was performed on the 8th day in positive control. The minimum edema volume was shown on the 32nd day of the treatment of dose 1 and dose of 3 ml SPF juice. The increasing or decreasing edema on different days of each treatment were affected by the metabolic state of rats. The higher the dose of SPF juice the decrease in edema volume. The positive control group (administered of Na-diclofenac) experienced death on day 10. This is caused by bleeding in the stomach due to continuous administration of drug delivery, so it indicates that natrium-diclofenac continuous usage for the long-term may cause side effect or toxic effect.  The effect of SPF juice on the average volume of rat foot edema is seen in Table 3.

 

Figure 3. Anti-Inflammatory of SPF Juice Against Edema Volume

Table 3. Volume Edema of Rats 

Groups	Mean of Edema Volume	Notation	Duncan	Inflammation (%)	Standard Deviation
Negative Control	1.789	c	0.140	78.9%	0.050
Normal Control	1.033	a	-	3.3%	0.057
1 ml SPF /200 gr BW	1.673	bc	0.134	67.3%	0.034
2 ml SPF /200 gr BW	1,.77	c	0.137	77.7%	0.062
3 ml SPF /200 gr BW	1.608	b	0.128	60.8%	0.113

 

Based on the result of the analysis of variance, it was found that SPF juice administration could decrease the edema volume on dose-dependent manner. The lowest mean volume of edema was performed in  3 ml SPF juice/200 BW which has an edema volume of 1.608 ml and inflammation percent of 60.8%. The highest mean edema volume was found in negative control treatment because of 0.1 ml CFA-induced rat without medication or SPF juice which average edema volume of 1.789 ml and inflammatory percent of 78.9%. The lower the average volume of edema and percent inflammation showed the anti-inflammatory potency is higher. This antiinflammatory activity is higher than ethanolic extract of Daturemetel Linn for decreasing hind paw edema volume with  dose 100 mg/200 Kg BW that decrease 45.6% in 5 hours [19] and Aqueous extract of Memecylonumbellatum root in dose 25 and 50 mg/200 Kg BW [20].Its indicate that 3 ml of SPF juice might inhibit the production of inflammatory mediators [17].

 Anti-inflammatory potency was found in SPF juice because of its antioxidant content. Galactomannan has the IC50 value of 22.109 mg/mL, which means that galactomannan concentration of 22.109 mg/mL is required to cause 50% inhibitory resistance to oxidation [2]. This is supported by Chen, et al [21], that galactomannan has a radical scavenging effect against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and superoxide. Therefore, inflammation can be inhibited by fighting radicals and inhibiting the secretion of inflammatory mediators and pain. Galactomannan is able to decrease the permeability of blood vessels of experimental animals thus inhibiting the occurrence of inflammatory mechanisms [18].

 

 In the study [22], Sugar palm fruit has an antioxidant activity of 37.74%. The antioxidant SPF juice is suspected to inhibit the formation of Reactive Oxygen Species (ROS) by CFAs that act as free radicals. CFA can lead to oxidation in the injected area, in the presence of oxidation-resistant SPF juice can be prevented because antioxidants donate hydrogen atoms to replace the released electrons.

 

 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. Therefore, treating arthritis with plant-derived compounds which are accessible and do not require laborious pharmaceutical synthesis seems highly attractive [23]. The mechanism of antioxidant as anti-inflammatory of SPF juice is almost identical to the anti-inflammatory mechanism of NSAIDs (Non-Steroid Anti Inflammatory Drugs), ie through cyclooxygenase (COX) pathways.  Cycloxygenases (COX) are the principle enzymes involved in the metabolism of arachidonic acid generating prostaglandins which are the genuine mediators of inflammation and pain [24]. Antioxidants in SPF juice are safer because in this study it was found that treatment with NSAIDs in the form of natrium diclofenac resulted in gastric bleeding, whereas the oral treatment of SPF juice did not showed the same effect. NSAID's works by inhibiting prostaglandin produced by COX-1 as a gastroprotective and pro-inflamatory prostaglandin produced by COX-2 (induced in inflammatory cells). Inhibition to COX-1 can cause gastric ulcers and increased risk of bleeding [11, 25]. It became the basis of the allegation that antioxidants and anti-inflammation in SPF juice inhibit COX-2 without inhibiting COX-1. Therefore, antioxidant and anti-inflammation of SPF juice can prevent the occurrence of pain and inflammation. In addition to galactomannan and antioxidants there are other compounds found in SPF like fiber, protein, fat, calcium, phosphorus, iron, vitamin A and B vitamins that are thought to have an effect on analgesic and inflammation.

 

CONCLUSION:

The effect of SPF juice consumption for pain relief on experimental rats induced by acetic acid gave significantly different results. The best treatment was shown in the dosage treatment of 3 ml/200 gr BW of SPF juice with the average amount of 3,190 or the analgesic power of 69.7%. The effect of SPF juice intake for decreasing edema in experimental rats induced by CFA gave significantly different results. The best treatment was shown in 3 ml/200 gr BW of SPF juice with an edema volume of 1,608 ml compared to the control that has 1.789 ml of edema. So that, SPF juice has the potency to developed as anti-analgesic and anti-inflammatory for osteoarthritis syndrome.

 

ACKNOWLEDGEMENT:

The authors would like to thanks to Indonesian Goverment through the minister of Research and Technology and higher education for grant 2016.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Accepted on 05.06.2018           © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(9): 3745-3751.