1. INTRODUCTION:

Inflammation is one of the underlying cautious reactions of our regular opposition framework to tainting or cell harm. The inflammatory reaction always participates in an significant part of host defense, although it can lead toward pathogenesis of many diseases such as like Cancer, Cardiovascular Dysfunction, Rheumatoid Arthritis etc^1-6. The majority of these anti-inflammatory drugs inhibit synthesis of prostaglandins, therefore interfere in inflammatory flow⁷. However, their habitual or excessive utilization causes side effects like gastric ulcer, renal failure, liver toxicity et^8-9. In these circumstances, use of herbal drugs to treat inflammation with connected situation become feasible.

Kashayam (Kwath/Kashaya) is a kind of oral Ayurvedic formulation prepared by boiling herbal ingredients with water.

Kokilaksha Kashayam is one such polyherbal formulation traditionally used to treat pain and inflammatory disorder^10,11,12. The formulation contains three common Ayurvedic herbs namely Asteracantha Longifolia, Tinospora Cordifolia and Piper longum (Table 1). The scientific justification given in favor of the claimed activities of this formulation has not been established yet. Moreover, the variation in quality between marketed formulations may lead to safety and efficacy issues. Current research work is therefore intended to validate anti-inflammatory and analgesic effects of in-house Kokilaksha Kashayam formulation (KKT) by various animal models and its activity was compared with the marketed Kokilaksha Kashayam formulation (KKM).

2. MATERIALS AND METHODS:

2.1 Preparation of Kokilaksha Kashayam:

The ingredients of Kokilaksha Kashayam were collected from a local Market of Mysore, India. They were authenticated by Natural remedies, Bangalore, India. The dried ingredients were crushed to the form fine powder and mixed well. The formulation was boiled with sufficient water in a low flame up to one-eighth of the initial amount the process called Decoction, cooled and stored in refrigerator till administered^13,14. The marketed formulation used for comparison was manufactured by The Arya Vaidya Pharmacy Limited (Batch No-AGL 174).

Table: 1 Ingredients of Kokilaksha Kashayam

S. No	Scientific name	Common name	Used Part(s)	Quantity (gms) used
1.	Asteracantha Longifolia	Kulikhara, Kokilaksah	Dried seeds	25grms
2.	Tinospora cordifolia	Heart-leaved moonseed, Guduchi	Dried stems	25 grms
3.	Piper longum (Adjuvant)	Indian long pepper,pipli	Dried Fruits	50 mg

2.2 Animals:

Wistar albino rats (150-200 g) and Swiss albino mice (30-40 g) of either sex were utilized in the study. All the experimental protocols were approved by the institutional animal ethics committee^15-18.

2.2.1. Evaluation of anti-inflammatory activity:

The Albino rats were divided into eight groups of 6 each as per the following:

Group I: Control group, received normal saline (10 ml/kg, p.o.)

Group II: Orally received 125mg/kg of KKT

Group III: Orally received 250mg/kg of KKT

Group IV: Orally received 500mg/kg of KKT

Group V: Orally received 125mg/kg of KKM

Group VI: Orally received 250mg/kg of KKM

Group VII: Orally received 500mg/kg of KKM

Group VIII: Orally received 1mg/kg of Indomethacin

Edema was induced in rats by injecting 0.1ml of 1% W/V of carrageenan on the sub-planter area of the right hind paw of experimental rats. The of paw volume of each rat was calculated 1 hour previous to carrageenan injection introduced and at 0min, 15min, 30min, 60min, 120 min and 180 min after the carrageenan injection introduced by utilizing plethysmometer.^19-22

2.2.2 Evaluation of analgesic activity:

2.2.2.1Eddy’s hot plate method:

The experiment was performed in Eddy’s hot plate method per standard procedure^23,24. Swiss albino mice were divided into eight groups of six animals and dosing was done as per the following:

Group I: Control group, received normal saline (10 ml/kg, p.o.)

Group II: Orally received 125mg/kg of KKT

Group III: Orally received 250mg/kg of KKT

Group IV: Orally received 500mg/kg of KKT

Group V: Orally received 125mg/kg of KKM

Group VI: Orally received 250mg/kg of KKM

Group VII: Orally received 500mg/kg of KKM

Group VIII: Orally received 40mg/kg of Tramadol

The cut off latency times were calculated for every animal.

2.2.3 Analgesic activity evaluation by tail flick method:

The Tail flick method was perform as per standard method²⁵ with the minor modifications done by utilizing a plantar test tool (7370 plantar test) with excessive powerful IR emission (strength of IR was 99) through the cut-off point was 5 seconds. The experimental animals were divided into eight various groups with the mean basal latency time was 2.0+0.4 seconds. Every experimental animals groups were treated orally with vehicle or KKT and KKM. The time of latency was recorded on 0, 30, 60, 120 and 180 minutes time intervals after the administration of test and standard drugs.

2.2.4. Evaluation antinociceptive pain by formalin induced model:

Evaluate the chemical-induced severe along with boosting pain and it was follow as per Hunskaar and Hole. Each group contain six animals, were treat with vehicle or KKT and KKM like following was 125, 250 and finally 500mg/kg, p.o. and Indomethacin was 10mg/kg, p.o.,1 hour before introduced 1% formalin solution used as an injection of 20μL in standard saline solution (v/v). Instantly following administration of formalin as an injection in the area of dorsal hind paw, then the pain response time noted, the animals remain licking or biting the paw through 1^stphase, 0 to 5 minutes time intervals and the 2^nd phase, 20 to 30 min for the experiment.

2.2.5. Writhing test by acetic acid induced model:

Test as per reported procedure²⁶, eight groups of the experimental Animals (n=6). 1 hour later than administration of 0.25% Na-CMC or KKT, KKM and Standard as like same as before, each and every experimental animals were received Acetic Acid 0.6% (0.1mL/10g) intra-peritoneal injections of designed intended for introduction of writhing. Then10 minutes after of introduced Acetic Acid injection, the figures of writhing was noted for 20 minutes interval designed for every session.

2.3. Statistical analysis:

All the experimental values were expressed as mean ± standard deviation. The results were analyzed by one-way analysis of variance (ANOVA) followed by a posthoc Tukey HSD test.

Fig. 1: The result of vehicle, Kokilaksha Kashayam (KKT and KKM, 125,250 and 500mg/kg) and Tramadol (40mg/kg) for antinociceptive property by using the hot plate method. Values within the outcomes be articulated by Mean+SEM, (n=6).

Fig. 2: The results of vehicle, Kokilaksha Kashayam (KKT and KKM, 125,250 and 500mg/kg) and Tramadol (40mg/kg) for antinociceptive property by using the tail flick method. Values within the outcomes be articulated by Mean+SEM, (n=6).

Fig. 3: The result of Kokilaksha Kashayam (KKT and KKM 125,250 and 500mg/kg) and Indomethacin (10mg/kg) for antinociceptive produce by formalin test are represent by A: first or Early phase and B: second or Late phase. Data expressed in the outcomes were articulated by Mean ± SEM, (n=6).

Fig. 4: The result of vehicle, Kokilaksha Kashayam (KK and KKM 125, 250 and 500mg/kg) and Indomethacin (10mg/kg) for antinociceptive effect by means of writhing test. Data in the outcoms are articulated by Mean ± SEM, (n=6).

Table 2: Kokilaksha Kashayam activity on Carrageenan induced paw edema model

Treatment	Given Dose (mg/kg)	Mean Paw Volume (ml)						% Inhibit ion after 180
		Time (Minutes)
		0	15	30	60	120	180
Control	Normal saline	0.77±0.30	1.05±0.16	1.43±0.12	1.63±0.17	1.80±0.19	1.72±0.02
KKT-I	125	0.76±0.12	1.18±0.18	1.52±0.32	1.27±0.15*	0.99±0.13**	0.69±0.40**	60
KKT-II	250	0.72 ±0.19	0.99±0.13**	1.16±0.72**	1.02±0.14**	0.86±0.16**	0.57±0.20**	67
KKT-III	500	0.71±0.23	0.82±0.20***	0.91±0.28**	0.80±0.54**	0.62±0.26**	0.45±0.30**	74
KKM-I	125	0.77±0.22	1.19±0.22	1.55±0.12	1.46±0.19*	1.12±0.22**	0.99±0.23**	43
KKM-II	250	0.75±0.26	1.12±0.13*	1.53±0.12	1.32±0.23**	0.97±0.16**	0.72±0.12**	58
KKM-III	500	0.73±0.02	0.98± 0.23**	0.92±0.16**	0.89±0.26**	0.72±0.21**	0.63±0.04**	63
Indomethacin	10	0.76±0.06	0.79±0.26***	0.72±0.21**	0.70±0.26 **	0.50±0.20**	0.46±0.09**	73

Effect of vehicle, Kokilaksha Kashayam (KKT and KKM 125,250 and 500mg/kg) and Indomethacin dose was 10mg/kg on paw edema of carrageenan induced model. data’s in the outcomes were expressed by Mean +SEM, (n=6).*p < 0.05, **p < 0.01appreciably different in comparison to vehicle control at respective time points.

3. RESULTS AND DISCUSSION:

The actual focus of current research was scientifically establishing the pharmacological response of Kokilaksha Kashayam. That work was considered to estimate Kokilaksha Kashayam's anti-inflammatory and anti-nociceptive pain prospective by utilizing various in-vivo pharmacological models, here comparison done with marketed formulation.

3.1. Kokilaksha Kashayam’s in vivo anti-inflammatory activity:

Anti-inflammatory activity of KKT and KKM was estimated by paw edema, carrageenan induced model (Table-2). The intraplantar injection (i.pl) of carrageenan in vehicle control rats led to a time reliant enhance the volume of paw that was maximum at 2 hours followed by subsidiary reduce at 3 hour. Animals were treated oral dosage of KKT and KKM at 125, 250, 500mg/kg and expressed considerable reduce the edema on paw at 3 hour, while observed higher dose 500mg/kg exhibited appreciable reduce the volume of paw for both KKT and KKM at 2 and 3 hours. but the activity of 125mg/kg dose was does not considerable different as compared with higher dose 500mg/kg. Additional, higher doses of KKT and KKM established highest anti-inflammatory property (records not expressed here) as 0.62+0.26 and 0.72±0.21 at 2 hour, followed by gradually decreased up to 3 hour. Standard anti-inflammatory drug Indomethacin at concentration of 10mg/kg expressed conspicuous anti-inflammatory property (>50%) at entire time duration but apart from up to 30min (0.72± 0.21)^27-31.

3.2. Kokilaksha kashayam’s antinociceptive activity:

3.2.1. Hot plate test’s evaluation:

KKT and KKM was assessing intended the antinociceptive pain achieved by utilizing hot plate test is exhibit in Fig. 1. And Control animals exhibited does not considerable variation in the latency time at various time intervals as differentiate with basal output signal for (0 min). KKT and KKM at the dose of 250mg/kg displayed considerable enhance during latency time on 30 and 120 min as differentiate with basal latency and higher dose 500mg/kg expressed appreciable increase in latency time at 120 and 180 min and differentiate with basal latency. % MPE (record not expressed here) exposed by the higher dose of KKT and KKT was established to be maximum (> 50%) at 120 and 180 min. usual opoid analgesic drug TMD at 40mg/kg; used intraperitoneally expressed evident enhance in latency at 60, 120 and 180 min. The antinociceptive pain property exposed by the TMD was established to be> 50% at entire time duration. Significantly, % MPE of KKT and KKM of higher dose was established to be alike with the TMD at 120 min (record not expressed here).^32-35.

3.2.2 Tail flick test’s evaluation:

Antinociceptive, pain consequence of Kokilaksha Kashayam was deliberate by mostly utilizing the spinally mediated tail flick retention time (Fig 2). Data propose, KKT and KKM at 250mg/kg displayed conspicuous enhance in latency time at 120 min duration as differentiate with basal retention time. Nevertheless, KKT and KKM at 500mg/kg expressed considerable antinociceptive activity by enhance in latency time at 60, 120 and 180 min with MPE (record expressed here) as 66.0±0.2% and 60.0±1.4% at 120 min. nevertheless, TMD expressed slow but surely increase in % MPE of antinociceptive pain conceivable up to 120 min. At The end outcome of the study evidently signify the KKT and KKM's significant antinociceptive pain activity perhaps arbitrate by spinal mechanism^.36

3.2.3 Formalin test’s evaluation:

Figure 3 shows the formalin test. The activity of mice with Kokilaksha Kashayam outcomes in an inhibition of formalin-induced in the test procedure. In near the beginning phase, the highest analgesia was observed at the dose of 500mg/kg of Kokilaksha Kashayam (KKT and KKM) (𝑃<0.01 with compared to control group) and that was equal to INDOMETHACIN in dose of 10mg/kg. In the delayed phase, eaxact the dose 250 mg/kg of Kokilaksha Kashayam (KKT and KKM) showed the highest analgesic effects (𝑃< 0.05 compared with control group) and that was comparable with Standard drug.³⁷

3.2.4. Writhing evaluation:

Anti-nociceptive pain, results of KKT and KKM alongside the visceral pain estimated by writhing test, the acetic acid induced. Normal control experimental animals expressed maximal writhing reaction 76.2+0.4 induced Acetic Acid 0.6% solution in normal saline by Intraperitoneal injection (Fig.4). After that orally dispense KKT and KKM on 125, 250 and 500mg/kg expressed considerable and dose response antinociceptive activity like KKT [86.0±0.8% (writhes: 10.6±1.2), 88.71±1.6% (writhes: 8.6±2.1), 90.55+3.2% (writhes: 7.2±0.6)] and KKM [85.56±1.8% (writhes: 11.0±0.6), 87.92±2.0% (writhes: 9.2±1.2), 89.76±2.6% (writhes: 7.8±0.6)] in that order. The usual NSAID drug, Indomethacin also generated significant reduce in the number of writhes (22.8 0±0.4) with 70.0 0±1.8% antinociceptive pain activity (record not shown here).^38,39

4. DISCUSSION:

The in-vivo experiment that assured the anti-inflammatory activity of Kokilaksha Kashayam in experimental rat, with inflammation models. Paw edema model is extensively utilized for anti-inflammatory property of a variety of formulations⁵ Normally this is a two phase event, throughout the first or inflammation early phase 0 to 2 hours intermediaries like histamine, 5-hydroxytryptamine and bradykinin acts a significant part, whereas throughout the second or accelerating late phase (post 2 hours) here raised the bio-synthesis of PGs and COX-2 also^22,40Kokilaksha Kashayam introduced on low doses was established to be operative state at the second or late phase. Nevertheless, at the high doses of Kokilaksha Kashayam exhibit significant reducing in both first and second phase of inflammation produced through carrageenan by reducing the discharge of a variety of inflammatory intermediaries interrupting on cyclooxygenase path. Kokilaksha Kashayam was assessed by hot plate, tail flick methods to establish its antinociceptive activity through supraspinal and spinal systems correspondingly⁴¹. After the oral administration of Kokilaksha Kashayam exhibited a dose reliant, considerable on anti-nociceptive pain related action in hot plate method, symptomatic of action be arbitrated by supraspinal system.

Tail flick method, Kokilaksha Kashayam exhibited significant antinociceptive property at higher doses. Parallel to standard drug TMD exhibited significant decrease in tail flick latency time duration and authenticate the analgesic prospective. Exact outcome proposes Antinociceptive action for Kokilaksha Kashayam overall necessitate both types supraspinal and spinal system. The Acetic acid persuade writhing instinctive it’s replica of visceral pain, useful method as pharmacological screening of peripherally exhibiting drugs for analgesic response^1,7,42. The statistics of Writhing test highlighted considerable peripheral anti-nociceptive action for Kokilaksha Kashayam through decreasing writhing reaction. As an excellent NSAID, Indomethacin as a dose 10mg/kg also expressed an important antinociceptive action, recognized to alleviate the pain reaction peripherally by the reducing the formation of prostaglandins, thromboxane by performing on the cyclooxygenase enzymes. The formalin induced model differentiates pain interested in first and second phases. It’s very helpful never only for evaluating the analgesic remedy but also expressing the exact mechanism of analgesia ^[25]^.The early or first phase responsible for neuropathic pain and second or late phase responsible for inflammatory pain arbitrate by prostaglandin and cytokines corresponding IL-1β, IL-6, TNF-α, eicosanoids and Nitric oxide^43,44. Although, Kokilaksha Kashayam expressed minimal response in the early or first phase at higher doses, it expressed outstanding response in the late or second phase at both dosage.

5. CONCLUSION:

The outcomes expressed in this experimental research work, were summarized that the Oral Polyherbal Ayurvedic Formulation ‘Kokilaksha Kashayam’ exhibit considerable anti-inflammatory along with analgesic proper, given that effective along with capable herbal substitute to presently obtainable NSAIDs further more here we studied two types of Kokilaksha Kashayam one prepared as per API in our laboratory and another marketed formulation, in our ayurvedic formulation as per API showing more potent than marketed formulation where both are same formulation but as per API we used fresh formulation and used Piper longum (Adjuvant ) as activity enhancer in our laboratory formulation. Our research expressed that Kokilaksha Kashayam 's Anti-inflammatory activities might attacking Arachidonic Acid flow and accord Pro-inflammatory cytokines.

6. ACKNOWLEDGMENTS:

All Authors are grateful to Sarada Vilas College of Pharmacy, Mysore to accomplish the Research process and the Author also especially grateful to Biju Patnaik University of Technology, Odisha as a registered research scholar.

7. ETHICAL CLEARANCE:

Human Rights:

This Research work not contains any Human studies by the any of the authors.

Animal rights:

National and Institutional all guidelines were followed for the care and use of laboratory animals and previous Ethical permission taken.

8. CONFLICT OF INTEREST:

Authors are declaring from best of the knowledge and expressed, they don’t have any conflicts of interest.

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Received on 24.08.2020 Modified on 05.06.2021

Research J. Pharm. and Tech. 2022; 15(5):2255-2260.

DOI: 10.52711/0974-360X.2022.00375