INTRODUCTION:

Fever is one of the oldest clinical disorders and a leading cause of hospitalisation worldwide¹. Ḥummā (Fever) is considered a systemic disease in Unani system of medicine (USM) and defined as “aberrant body heat that spreads through veins from the heart to other organs”.

This atypical rise in body temperature strongly impacts the functioning of vital organs of the body². Pyrexia is designated with the increased body temperature corresponding to augmentation of the hypothalamic set point. These elevations are viewed as a source of discomfort³.

Pain is considered an indication of disease or a signal for infection in the body that guided a common person to consult a physician for the same. Task force on the taxonomy of the IASP has defined pain as: “An unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage”⁴. According to the USM, Pain is also Ghayr Tabi‘i (abnormal or unnatural) condition of the human body and defined as “a perception of incongruity in the body”⁵. It is evident that herbal medicines have been used for ages to cure a variety of diseases. Hence the development and evaluation of safer analgesics and antipyretics are still in progress. Therefore, this study was carried out to evaluate the analgesic and antipyretic effect of polyherbal Unani formulation (PUF) in animals. This classical PUF has been used to cure fever and pain for a long.

MATERIALS AND METHODS:

Crude drugs collection and Authentication:

The crude ingredients of PUF were acquired from local retailers in Hyderabad. The purchased herbs samples were identified and verified by the Research Officer In-Charge Survey of Medicinal Plants (SMP) Unit of the NRIUMSD, Hyderabad. The voucher specimens were preserved at the SMP unit for records and future reference for Chiraita Shireen, Gilo-e-Sabz, Gul-e-Ghafis, Gul-e-Nilofar, Beikh-e-Kasni, and Khaksi as Voucher No. SMPU/CRI-Hyd 14115, 14116, 14117, 14118, 14119, and 14120 respectively.

Composition of PUF:

As per the classical Unani literature⁶ the composition of PUF is mentioned below in Table 1.

Test drug preparation and dosage:

The test drug was formulated as a decoction (Joshanda) freshly prepared on daily basis. All the ingredients were transformed into coarse powder and mixed to make the quantity of 18gm. The coarse powder of drugs was soaked in 180ml of water overnight. It was boiled in the morning till the quantity remained half. After straining, the decoction was adjusted to 90mL, the daily human dose⁷. The decoction was orally administered to animals. Test doses of PUF in this study are represented in mg/kg (rather than ml/kg) for convenience. The percentage yield of the prepared decoction was calculated by converting the prepared decoction into solid powder by using a freeze drier (Lyophilizer). After lyophilization of 90mL (daily human dose) of decoction, the yield of dried extract was ≈1.40g (i.e., 1400mg) which was considered as the daily adult dose. The human adult dose was converted to 144mg/kg (rounded to 150mg/kg bw) for rats using the body surface area conversion method⁸. Similarly, the equivalent dose for the mouse was obtained as 288mg/kg bw which was rounded to 300mg/kg bw. A second dose i.e., a double dose (twice of HED) of 300mg/kg bw/day for rats and 600mg/kg bw/day for mice, respectively, to study the dose-dependent effect of the test drug.

Chemicals and Reagents:

Brewer’s yeast, Acetic acid, Paracetamol, Diclofenac sodium, Buprenorphine, etc.

Animals:

Sprague Dawley (SD) albino male rats (8-10 weeks) and Swiss albino mice (6-8 weeks) were procured from NIN, Hyderabad for this study. Rats and mice were accommodated in polypropylene cages in a group of 6 animals in each cage. They were housed to maintain the temperature and humidity as per CPCSEA guidelines and habituated to laboratory conditions for 7 days before their use. The periodic measurement of body weight was done. The Local Institutional Animal Ethics Committee (IAEC) approved the study protocol on 18/12/2018 (CRIUM/IAEC/2018/02/P04).

Antipyretic activity:

Yeast-Induced Pyrexia:

The experiment was conducted on male Sprague-Dawley rats. The animals were categorized into four groups, each including six rats. Originally, the standard body temperature of animals was measured using a rectal thermometer. To produce pyrexia, a 10ml/kg saline combination with 15% Brewer's yeast was injected below the nape. Following the delivery of yeast, food was promptly withheld. The body temperature was measured by inserting a digital thermometer 2 cm into the rectum for 60seconds. Rectal temperature was measured after 18hours of injection. The study included animals that had a minimum increase in body temperature of 0.5ºC. According to the respective group, the animals were given normal saline, the standard medicine (Paracetamol), and the test formulation (PUF) through oral administration. Rectal temperatures were measured again at 30, 60, 120, and 180 minutes after administering the dose.

The details of the groups are as follows:

Group-I (Normal Control): After 18hours of S.C. yeast injection, animals in this group were given 0.9% w/v Normal saline, orally.

Table 1: Ingredients of PUF.

S. No	Name	Botanical Name	Part used	Quantity
1.	Gul-e-Ghafis	Gentiana olivieri Griseb.	Flower	3 gm
2.	Gul-e-Neelofar	Nymphaea nouchali Burm. F.	Flower	3 gm
3.	Chiraita Shireen	Swertia chirayita (Roxb) H.Karst.	Whole herb	3 gm
4.	Beikh-e-Kasni	Cichorium intybus L.	Root	3 gm
5.	Khaksi	Sisymbrium irio L.	Seeds	3 gm
6.	Gilo-e-Sabz	Tinospora cordifolia (Willd.) Miers ex Hook.f. & Thomson	Stem	3 gm

Group-II (Standard Control): After 18hours of S.C. yeast injection, animals in this group were given with 70 mg/kg bw of Paracetamol per orally.

Group-III (PUF-150): After 18hours of S.C. yeast injection, this test group was given PUF 150mg/kg orally.

Group-IV (PUF-300): After 18hours of S.C. yeast injection, this test group was given 300mg/kg PUF per orally.

Analgesic activity:

(I) Acetic Acid-Induced Writhing Test:

This model was executed using the methods proposed by Whitkin in 1962. A total of six Swiss albino mice were assigned to each of the four groups. These groups are categorised as follows.

Group-I (Normal Control): Animals in this group were treated with 0.9% w/v normal saline per orally.

Group-II (Diclofenac Sodium): Animals in this group were treated with Diclofenac sodium (15mg/kg) orally.

Group-III (PUF-300): PUF at 300mg/kg bw was given orally.

Group-IV (PUF-600): PUF at 600mg/kg bw was given orally.

Each mouse received an IP injection of 0.6% acetic acid (10ml/kg) 30 minutes after the administration of the respective treatment. After 5minutes of injection, the number of writhing (hind limb stretching) was counted for 20 minutes^12,13.

The percentage inhibition was calculated using the following formula:

% Inhibition =	{ (NWc -NWt) × 100 }
	NWc

Where

NWc = No. of writhes in the Control group

NW_t = No. of writhes in the treatment group

(II) Eddy’s Hot Plate Test:

Eddy et al., 1950's analgesic effect approach was used. The following four groups of six male SD rats were formed.

Group-I (Normal Control):

Rats were given 0.9% w/v NS orally.

Group-II (Buprenorphine): Rats in this group were given buprenorphine injection of 0.1mg/kg subcutaneously.

Group-III (PUF-150): Rats were given PUF at the dose of 300mg/kg bw orally.

Group-IV (PUF-300): Rats were given PUF at the dose of 600mg/kg bw orally.

Each group was administered their respective therapies following a 12hour period of fasting. The animals were placed on a hot plate (Analgesiometer) that was maintained at a temperature of 55°C in order to evaluate the latency period. Analgesia was defined as an increase in the time it takes for a response to pain, such as licking or flicking of the hind limb or jumping. The latency time was assessed at 0, 30, 60, 90, and 120 minutes after the administration of therapy in their respective groups. To prevent tissue harm, a time limit of 20 seconds was implemented. The Latency, quantified in seconds, denotes the duration it took for the rats to react to the heat stimulus, commonly by leaping¹¹.

RESULTS:

Antipyretic effect of PUF on yeast-induced pyrexia in rats:

There was an approximate rising of 1⁰C rectal temperature in all treatment groups after 18hours of subcutaneous yeast injection as shown in Table 2. Oral administration of paracetamol at the dose of 70mg/kg bw (Group II) caused a significant (p<0.001) reduction in rectal temperature at a time point of 60 and 120 minutes after treatment as compared to normal control (Group I). PUF -150mg/kg bw (Group III) did not show any significant reduction in rectal temperature as compared to normal control and paracetamol control at any time point tested. However, PUF-300mg/kg (Group IV) caused a significant (p<0.001) reduction in rectal temperature at 60 minutes after drug administration as compared to the normal control. The result is shown in Table 2 and Figure-1 (Graph).

Table 2: Effect of PUF on the rectal temperature

Treatment	Rectal Temperature (⁰C)
	Basal	After 18 hours of Yeast injection	After treatment
	Basal	After 18 hours of Yeast injection	30 min	60 min	120 min	180 min	240 min
Normal Control (NS 0.9% w/v)	35.67 ±0.32	36.85 ± 0.12	36.42± 0.24	37.17± 0.32	37.08 ± 0.27	36.13± 0.40	36.37± 0.34
Paracetamol (70mg/kg)	35.28 ±0.32	36.50 ± 0.27	35.42± 0.320	34.55±0.14***	34.92±0.31***	34.85± 0.29	35.55± 0.20
PUF (150 mg/kg)	35.12±0.35	36.22±0.32	36.05 ± 0.24	36.00 ± 0.38	36.18 ± 0.39	36.07± 0.39	35.68 ± 0.07
PUF (300 mg/kg)	35.00 ±0.25	36.08 ± 0.22	35.30 ±0.38	35.05± 0.31***	35.85 ± 0.28	35.70± 0.46	36.22± 0.10

Each value signifies Mean ± SEM, n = 6. Statistical significance by ANOVA, ***P <0.001 vs normal control.

Analgesic activity of PUF on acetic acid-induced writhing response in mice:

Prior to the injection of acetic acid, none of the treatment groups exhibited any indications of abdominal constrictions, such as a writhing response. After injecting acetic acid, writhes were detected in Group I, which served as the usual control, 20 minutes later. The average number of writhes was 21.33±3.82. In Group II, Diclofenac was administered orally at a dose of 15 mg/kg bw. This resulted in a substantial (p<0.05) decrease in the number of writhes (9.00±1.6), representing a 57.81% inhibition compared to the normal control group.

In groups III and IV, the test medication was given at doses of 300 and 600mg/kg bw, respectively. The number of writhes seen in these groups was 13.50±2.59 and 6.67±0.88, respectively. Following PUF-300mg/kg administration, writhes decreased by 13.50±2.59%. This number was not statistically significant compared to the normal control group. The administration of PUF-600 mg/kg bw (Group IV) resulted in a noteworthy decrease (p<0.01) in abdominal constrictions and stretching of hind limbs. The observed number of writhes (6.67± 0.88) was decreased, resulting in a 68.71% inhibition compared to the usual control. The positive control group given Diclofenac and the experimental medication PUF showed no noticeable difference. The observations are shown in Table 3 and Figure 2 (graph).

Table 3: Effect of PUF on acetic acid-induced writhing in mice

Treatment	No of writhing	Inhibition (%)
Normal Control (N.S. 0.9% w/v)	21.33±3.82	-
Diclofenac 15mg/kg	9.00±1.60*	57.81
PUF - 300mg/kg	13.50±2.59	36.71
PUF - 600mg/kg	6.67±0.88**	68.71

Values presented as Mean±SEM; n=06; One way ANOVA; *=p<0.05 vs normal control, **= p<0.01 vs normal control

Analgesic activity of PUF on Eddy’s hot plate test:

There was no significant difference in the latency response (paw withdrawal) among any of the groups before treatment. Administering buprenorphine subcutaneously at a dosage of 0.1mg/kg bw (Group II) significantly increased (p<0.05) the time it took for the paw to withdraw in response to a thermal stimulation, 30 minutes after treatment. This demonstrates an analgesic effect in comparison to the control group (Group I). Group III, administered the test medication PUF orally at a dosage of 150mg/kg bw, showed significant analgesic effectiveness. This was demonstrated by a statistically significant increase (p<0.05) in the time it took for a response to occur at 90 minutes and 120 minutes after the administration of the treatment, when compared to the control group without any drug (Group I). Furthermore, Group IV, which was administered the test medication PUF orally at a dosage of 300mg/kg bw, demonstrated an analgesic effect 120minutes after receiving the drug. This led to a significant (p<0.01) increase in latency time when compared to the usual control group (Group I). The result is shown in Table 4 and Fig 3.

DISCUSSION:

Anti-pyretic Activity:

Pyrexia is commonly observed as a subsequent consequence in cases of infection and other inflammatory illnesses. An elevated body temperature is mostly caused by an increase in the level of prostaglandin (PG). Inflammatory conditions involve a pathophysiological process that results in the production of pro-inflammatory mediators, such as cytokines (including interleukin-α, and TNF-α). These mediators cause an increase in prostaglandin-E2 (PGE2) levels near the hypothalamus, which in turn stimulates the hypothalamus to raise body temperature. In a similar manner, Brewer's yeast was found to stimulate the production of both TNF-α and prostaglandins. Antipyretic drugs work by inhibiting the increased amounts of PGE2, which in turn reduces the rising body temperature¹⁴.

One of the basic, cheap and reliable methods for screening antipyretic agents is the Yeast-induced pyrexia model in rats¹⁵. Therefore, it was applied for the screening of tested formulation PUF. There was no significant difference observed among all treated groups after 30min. of treatment. Paracetamol (70mg/kg) treated rats, at the time point of 60 and 120min showed significant (<0.001) reduction in rectal temperature as compared to normal control, confirming the validity of the model. PUF-150mg/kg treated animals showed no significant reduction in rectal temperature at any time point interval. PUF-300mg/kg treated animals showed a significant (<0.001) decrease in rectal temperature 60 minutes after medication administration compared to the normal control. At the time point of 120min, PUF-300mg/kg also reduced the rectal temperature as compared to normal but it was not statistically significant. No significant difference was observed in the reduction of rectal temperature in any group at 180 and 240min.

The study found that a higher dose level of PUF (300 mg/kg bw) had antipyretic effect in male SD rats. The findings of this investigation were similar to other research studies on the assessment of the fever-reducing benefits of a specific herbal medication in this particular formulation. PUF is a blend of various herbal components, each of which has been found to have anti-fever properties according to the literature.

Khan MA conducted a study to assess the fever-reducing effects of N. stellate on yeast-induced fever in Wistar rats and found evidence of its antipyretic action. The findings indicated that it led to a decrease in temperature by impacting the synthesis of prostaglandins, with a greater impact observed at larger doses¹⁶. The majority of the chemical constituents of N. stellate are similar to N. nouchali and are used as synonyms of each other. A compound nymphayol isolated from N. stellate showed significant antipyretic activity in experimental test models¹⁷. In a preclinical study, the whole plant of S. chirayita (Chiraita Shireen) extract showed significant antipyretic activity and was recommended as a potent antipyretic drug that can be used in fever¹⁸. Another efficacy study supports the antipyretic activity of one of the important constituents Khaksi in PUF. The ethanolic extract of S. irio (Khaksi) seeds exhibited significant antipyretic action. In another study, an aqueous extract of Khaksi showed a significant antipyretic effect in Wister rats¹⁹. Similarly, the water-soluble extracts of Gilo (T. cordifolia) exhibited a certain degree of antipyretic action, although it cannot be considered significant. It is possible that a greater dosage could have resulted in statistically significant benefits²⁰.

Based on the data collected in this study, it was found that administering PUF decoction at a dose of 300 mg/kg significantly lowered rectal temperature compared to the normal control after 60 minutes of treatment. PUF may have suppressed the activity of the cyclooxygenase enzyme. The cyclooxygenase enzyme is essential for the production of prostaglandins, which in turn helps to lower body temperature and alleviate fever. Nonsteroidal anti-inflammatory medicines (NSAIDs) are a type of medication that specifically inhibits the enzyme COX-2. They are also effective in reducing fever (antipyretics). Acetaminophen exhibits little inhibitory effects on cyclooxygenase in peripheral tissue. Nevertheless, the brain oxidises it through the cytochrome system p450, resulting in the inhibition of cyclooxygenase activity by its oxidised form. Furthermore, the antipyretic effect of paracetamol may be attributed to its inhibition of the COX-3 enzyme in the brain. PUF potentially decreased the level of PGE-2 in the hypothalamus via affecting the cyclooxygenase enzyme²¹.

Analgesic activity:

The pain-relieving effect of PUF was assessed by conducting the acetic acid-induced writhing test in Swiss albino mice (a peripheral model) and Eddy's hot plate test in rats (a central model). The Acetic acid-induced writhing test model stimulates a localised inflammatory response, resulting in the release of free arachidonic acid from the tissue phospholipid²². The administration of acetic acid into the peritoneal cavity caused a response marked by a sequential movement of narrowing and lengthening that spread towards the tail along the abdominal wall. Occasionally, the response was accompanied by trunk twisting and later followed by hind limb extension^23,24. This methodology is both economical and reliable in terms of its ability to accurately assess peripherally acting analgesics²⁵. The pain-relieving effectiveness in this animal is thought to be influenced by peritoneal mast cells, acid-sensing ion channels, and the prostaglandin pathways²⁶.

The present investigation demonstrates that PUF significantly decreases the frequency of abdominal writhing in Swiss albino mice. After 20 minutes of the test period, PUF-150mg/kg bw showed analgesic effect by decreasing the number of writhes by 36.71%, while PUF-300mg/kg bw exhibited the maximum analgesic activity by reducing the number of writhes by 68.71%. The administration of the conventional medication Diclofenac at a dosage of 15mg per kg of body weight resulted in a reduction in writhing episodes by 57.81%.

The results of this study showed that PUF has peripheral analgesic effects in a dose-dependent manner. The findings suggested that PUF could potentially exhibit comparable effects as traditional therapeutic medicines (NSAIDs) by diminishing pain perception in nociceptors through the inhibition of prostaglandin synthesis. These results align with earlier experimental investigations on the assessment of the pain-relieving properties of the herbal medicinal plant. Prior studies have assessed the analgesic effects of various individual drugs found in PUF. For instance, The methanol extract obtained from the N. nouchali flower exhibited strong and notable antinociceptive action in the acetic acid-induced writhing test. The extract demonstrated a reaction that varied according to the dosage, as the lower dosage (200mg/kg) resulted in the inhibition of 59.97% of writhing, while the higher dosage (400mg/kg) achieved a maximum inhibition of 64.75%. These results were similar to the effects of the reference medication diclofenac sodium³¹. The administration of the medicine Gilo (T. cordifolia) significantly decreased the number of writhes in comparison to the control group²⁷. An evaluation was performed to determine the pain-relieving properties of the ethanolic root extract of S. chirayita utilizing animal models. The effectiveness of the pain-relieving properties was evaluated in mice using the acetic acid-induced writhing test. The extract given at doses of 200 and 400mg/kg showed inhibitory effects of 41.76% (p<0.001) and 58.29% (p<0.001) on writhing, respectively. Animal studies have demonstrated significant analgesic effectiveness of Khaksi seeds (S. irio)²⁸.

Through a literature search, it is discovered that the presence of flavonoids, phenols, and tannins in herbs or plants is responsible for their analgesic activity^29,30. Among these compounds, flavonoids and saponins have been extensively studied and are recognised for their capacity to suppress pain perception. Flavonoids demonstrate anti-inflammatory characteristics by blocking enzymes involved in the production of the chemical mediator of inflammation³¹. The method by which PUF works is likely due to the presence of phytochemicals that can potentially inhibit peripheral pain mediators or arachidonic acid pathways, namely involving cyclooxygenase and/or lipoxygenases. Consequently, the transmission of pain is inhibited³².

Rats were utilised to evaluate the central analgesic effect of PUF by doing Eddy's hot plate test. This paradigm is specifically used to screen centrally acting medicines. The hot plate test is a frequently used method to assess central nociceptive activity. It assesses the intricate reaction to a sudden, non-inflammatory nociceptive stimulus^33,34. The Hot Plate Model quantifies the increase in pain threshold of rats and mice in response to heat. It is widely accepted that animals' reactions to uncomfortable heat stimuli in the hot plate method, such as licking their paws and jumping, are controlled by the parts of the nervous system located above the spinal cord^35,36. Moreover, any agent that prolongs the hot plate latency in this test must be exerting its effects in the central nervous system³⁷.

The analgesic effect of opiates, such as morphine and its derivatives, is a result of their interaction with several receptors found in both the spinal and supraspinal areas³⁸. Spinal analgesia seems to be controlled by μ2, d2, and κ1 receptors⁴². The analgesic effect of plant extracts in hot plate tests may be attributed to their interaction with several receptors found in supra-spinal locations. The analgesic and sedative effects of lactucin and its derivatives, lactucopicrin and 11ẞ 13-dihydrolactucin, were assessed in rats. These compounds are bitter sesquiterpene lactones found in Kasni (C. intybus). The compounds exhibited analgesic properties in the hot plate test. In a separate investigation, the analgesic activity of Gilo (an extract derived from T. Cordifolia) was assessed. The results showed that Gilo caused a statistically significant level of analgesia (p<0.05) when compared to the control group using the hot plate method. Thus, it is possible that PUF has elicited centrally-acting analgesic effects in the Hot plate test. Multiple research projects have confirmed the analgesic, anti-inflammatory, and antipyretic properties of natural herbs i.e. Cassia occidentalis L.³⁹, Tectona grandis L.^40, Nyctanthes arbor-tristis L.⁴¹, and Atylosia rugosa Wight and Arn.⁴². Preclinical studies have verified the analgesic and anti-inflammatory properties of Anthocephalus cadamba Miq.⁴³, Datura metel L. root⁴⁴, and the bark of Neolamarckia cadamba Roxb⁴⁵, the leaf extract of Ficus dalhousieae Miq. leaves⁴⁶, the leaves of Caesalpinia sappan L.⁴⁷, and Rotula aquatica Lour leaves⁴⁸.

This study demonstrated that in Eddy's hot plate model, there was no statistically significant disparity in the latency period among the experimental groups at the baseline. After a 30-minute treatment, a significant difference was observed in the latency duration of the standard control group that received buprenorphine (0.1 mg/kg s.c.), as compared to the normal control group. The administration of PUF-150mg/kg resulted in a statistically significant (p<0.05) prolongation of the time it took for a response to occur, specifically at the 90- and 120-minute marks following treatment. The administration of PUF-300mg/kg resulted in a statistically significant (p<0.01) increase in the latency duration at 120 minutes in the hot plate method. Unani treatments sometimes utilises combinations containing multiple herbs to effectively treat fever and other ailments. The test Unani formulation had significant antipyretic and analgesic effects. The test composition has been utilised for centuries to manage fever, and the investigation demonstrates its antipyretic effectiveness.

CONCLUSION:

The data from the current investigation demonstrated that PUF exhibits antipyretic effect when administered at high doses (300mg/kg bw) in the yeast-induced pyrexia model. Similarly, a greater dosage of PUF (300 mg/kg) demonstrated analgesic effects in the acetic acid-induced writhing test, resulting in a notable decrease in writhing movements. Significant analgesic effect was shown in Eddy's Hot Plate test at both dose levels of 150 and 300mg/kg. This preclinical study confirmed the ethnopharmacological efficacy of PUF in treating fever and pain.

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Received on 31.07.2023 Modified on 17.01.2024

Research J. Pharm. and Tech 2024; 17(7):3189-3196.

DOI: 10.52711/0974-360X.2024.00499

Treatment	Latency time of rat exposed to the hot plate (Sec)
	Before Treatment	After Treatment
	Before Treatment	30 min	60min	90min	120min
Normal Control (N.S:0.9%w/v)	7.28±0.436	6.80±0.668	4.95±0.628	5.18±0.450	4.20±0.416
Buprenorphine (0.1mg/kg)	6.25±0.099	12.07±0.099*	7.57±0.406	6.58±0.394	6.13±0.235
PUF (150mg/kg)	9.32±1.609	7.18±1.292	7.20±0.900	7.72±0.811*	7.52±1.309*
PUF (30mg/kg)	8.85±0.459	8.55±1.616	8.07±1.321	6.40±0.404	7.93±0.741**