Anti-Inflammatory Effect of an Ethanolic Extract of Ficus Dalhousiae Miq Whole Plant (EEFD) In Wistar Albino Rats

Natural goods include bioactive chemicals which have ability to become innovative medicinal treatments. During the past ten years, there was an increase in enthusiasm for plant-based medications, which have become a significant group for disease prevention. In accordance with the World Health Organization, over eighty per cent of individuals in poor countries rely on conventional medicines for basic health care, and a recent study found that over sixty per cent of patients utilize medicinal plants as part of their treatment ^[1]. Since tens of thousands of years ago, numerous individuals have used plants to heal a variety of illnesses. Various countries employ plants as medicine, and they are the foundation of a wide range of potentially strong medications. Conventional medical care has established a foothold in everyday life and is still the major source for medical care in the countryside of numerous countries that are developing. Compared to synthetic or contemporary drugs, these medications are generally inexpensive and safer. Because they are present in combination or in a pooled form of several molecules in the protoplasm of the plant cell, herbal compounds are less dangerous and can defeat the resistance established by infections ^[2]. The researchers discovered an intriguing species of Ficus of the family Moraceae thriving in rock fissures while doing botanical research in Kuntho Betta near Pandavapura in Mandya District (Karnataka State). Through careful inspection and a thorough search, this was identified as Ficus dalhousiae Miq. It’s notable that this extremely uncommon species is restricted to peninsular India. According to Wight’s collection from India, Miquel initially identified this species as Urostigma dalhousiae in 1847. However, in 1867, it changed its name to Ficus dalhousiae. Later, the species was discovered the Nilgiri Mountains in the altitudinal range ranging from 605-1,370 m by king (1888), Hooker (1890), Brandis (1906), and Gamble (1928). There have been sightings of this particular species in many places. The fact that there has only ever been one F. dalhousiae tree in the area confirms its rarity. This species can be found thriving on the tops of rocky hills in Savanadurga (Ramanagaram District of Karnataka) and Siddara Batta (Tumakur District of Karnataka). No matter where it grows, its range is relatively constrained within the extremely rare group of endangered plants ^[3]. Ficus dalhousiae Miq is a Moraceae plant. They’re exceptionally rare species seen in the Nilgiri highlands at elevations ranging from 605 to 1370 m. It is a 10 m tall tree with brown bark & simple oval leaves. The plant grows in damp deciduous woods and is found only in the Southern Western Ghats. It has a very limited number of inhabitants everywhere it expands which is presumably why it is listed as a vulnerable plant. Fruit is utilized as a cardio tonic, whereas leaves and bark are mostly employed in conditions of the liver and skin. The juice from leaves is anti dysentric in nature. Antispasmodic roots are used. According to tradition, bark can be used to cure high cholesterol levels and malignancy ^[4]. Our study was focused on novel anti-inflammatory medicines from natural botanical sources that may have low side effects was motivated by the serious adverse effects of steroidal and non-steroidal anti-inflammatory therapies ^[5].

MATERIALS AND METHODS

MATERIALS

The reagents used in qualitative analysis are Millon’s reagent, Ninhydrin reagent, Dragendroff’s reagent, Wagner’s reagent, benedict’ reagent, Fehling’s A and B solution, and the chemical used Ethanol (95%), Carboxymethyl cellulose (CMC), L-ascorbic acid, Disodium hydrogen phosphate, Potassium dihydrogen phosphate, Trichloroacetic acid (TCA), Ferric chloride, Hydrogen peroxide, Disodium phosphate, Monosodium phosphate, Tris HCl, Trypsin and chymotrypsin, Casein, Petchloric acid, and Diclofenac sodium.

Collection and authentication the plant

Ficus dalhousiae Miq was collected in its whole at Tirupati, Andhra Pradesh. The Ficus dalhousiae Miq. plant has been verified by Dr. K. Madhava Chetty, who has a number of advanced degrees and is an assistant professor in the field of botany. Sri Venkateswara College in Andhra Pradesh’s holy city of Tirupati.

Preparation of ethanolic plant extract

 The Ficus dalhousiae whole plant was washed with fresh water and dried in the dark at ambient temperature and in the shade to ensure cleanliness and hygiene. At room temperature, the brittle whole plant was grounded into powder using an electric grinder. The plant drug, whole coarsely powdered, is maintained in contact with the 95% ethanol in a sealed vessel for 6-7 days while being agitated frequently to disperse the soluble materials. The dried ethanolic extract of Ficus dalhousiae (EEFD) was stored in a day area in an airtight bottle for further studies ^[6].

Experimental animals

Ethical approval for the study (ID: CPCSEA/1657/IAEC/CMRCP/COL-22/106) was granted by the Institutional Animal Ethical Committee (IAEC) on November 9, 2022. The research included the use of 200-250 g adult male cross-bred albino rats. The animals had free access to pelleted food and running water in their plastic cages in a controlled environment (temperature: 28-31℃; photoperiod: roughly 12h sunlight per day; relatively humidity: 50-55%). Animals were dealt with only during cage cleaning and not at any other time except during experimental procedures. Animals were only employed as a last resort, and every attempt was made to lessen their suffering.

Treatment Groups

Group I-Normal Control: Normal saline (10ml/kg), p.o, OD for 28 days.

Group II-Toxic Control: Carrageenan (1%w/v), intraplantar injection to paw.

Group III-Standard Control: Diclofenac sodium (10mg/kg), i.p,

Group IV-Test Control (200mg/kg): EEFD (200mg/kg), p.o,

Group V-Test Control (400mg/kg): EEFD (400mg/kg),

Group VI- Test Control (800mg/kg): EEFD (800mg/kg).

Qualitative phytochemical analysis

The qualitative phytochemical analysis was performed to determine the presence of bioactive compounds like proteins, amino acids, alkaloids, flavonoids, tannins, phenolic compounds, carbohydrates, saponins, and coumarins ^{[7, 8, 9, 10]}.

In vitro Antioxidant Studies

Ferric Reducing Antioxidant Power Assay

Five different doses of EEFD (100, 200, 400, 800, & 1000 mg/ml) were mixed and L-ascorbic acid at the same concentrations, together with 2 ml of phosphate buffer (0.2 M, pH 6.6) and 2 ml of 1% potassium ferricyanide. The mixture was kept at 50^oC for 20 minutes. After adding 2 ml of 10% trichloroacetic acid (TCA), the mix was centrifuged at 1000 rpm for 10 minutes. The resultant 2 ml solution should be discarded and mixed with 2 ml of purified water and 1 ml of 0.1% ferric chloride (FeCl₃). Each experiment was repeated three times. Absorbance values were then measured using a UV-visible spectrophotometer at 700nm and recorded.

Hydrogen peroxide scavenging activity

Hydrogen peroxide was diluted to 20mM in a phosphate buffer of 50mM, pH 7.4. Each sample was diluted to a level of 0.1 ml in 50mM phosphate buffer pH 7.4 and put to the test tube. H₂O₂ absorbance at 230 nm was measured against a blank after 0.6 ml of H₂O₂ solution has been added and the tubes were shaken for 10 minutes ^[11].

In vitro Anti-inflammatory Methods

Inhibition of   Protein Denaturation

5 ml of the resulting solution encompassed 0.2 ml of fresh egg albumin, 2.8 ml of phosphate buffer saline (PBS, pH 6.4), and 2 ml of EEFD at doses of 10, 20, 40, 60, 80, and 100 mg/ml. Double-distilled water of the same volume was used as a control. After being heated to 70^oC for 5 minutes, the samples were left to incubate at 37^oC for 15 minutes. Once chilling was complete, the vehicle was used to measure their absorbance at 600 nm. The absorbance of diclofenac sodium was measured at concentrations of 10, 20, 40, 60, 80, and 100 mg/ml ^[12].

Proteinase inhibitory activity

Enzyme at a concentration of 0.5 ml, 25mM Tris HCl buffer at 1 ml, and test solutions at 10–100 g/ml, each in 1 ml. Incubation at 37^oC for 5 minutes was applied to the aforementioned mixture. Add 2 ml of 70% perchloric acid and boil at 70°C for 15 minutes with 1 ml of 0.8% w/v casein. The superannuated layer was separated from the rest of the mixture after centrifugation, and the absorbance at 276 nm was compared to that of a blank. Consequently, diclofenac sodium was selected as the gold standard ^[13].

In vivo Anti-inflammatory studies

Carrageenan-induced hind paw edema

Six groups of six male rats overall were randomly assigned. The rat’s weights ranged from 100 to 200g. An intradermal injection of carrageenan (0.05 ml, 1% w/v, normal saline solution) was injected into the plantar side of the left hind paw 1 hour after the administration of the vehicle, diclofenac (10 mg/kg), or EEFD (200, 400, 800 mg/kg). One, three, and six hours after receiving a carrageenan injection, the paw volume was measured utilising a plethysmometer.

RESULTS AND DISCUSSION

RESULTS

Table 1: Results of Qualitative phytochemical analysis

Here, + indicates positivity and - negativity.

An abundance of components, including amino acids, proteins, alkaloids, saponins, flavonoids, phenolic compounds, tannins, and triterpenoids, were found by qualitative phytochemical study of EEFD.

In vitro Antioxidant Studies

Ferric Reducing Antioxidant Power Assay

Table 4: Ferric Reducing Antioxidant Power Assay Results

The values are shown as a Mean ± SEM.

The present study shows a link between dose and activity within the designated 100-1000 ng/ml range of the plant F. dalhousiae. According to Table 4, EEFD's IC50 value was 170 ± 5.77µg/ml, whereas L-ascorbic acid's was 8.71 ± 1.06 µg/ml.

Hydrogen peroxide scavenging activity

Table 5: Hydrogen peroxide Scavenging Activity Results

Values are represented as Mean ± SEM

Within a concentration range of 100-1000 μg/ml, several parts of F. dalhousiae were shown to have scavenging action against hydrogen peroxide. With an IC50 of 302.2 ± 22.1μg/ml, the EEFD demonstrated potent H₂O₂ scavenging activity. The scavenging activity of standard L-ascorbic acid against H₂O₂ was 246.0±19.4μg/ml, in contrast. According to Table 5, the H2O2 inhibition rates of F. dalhousiae plant extract and L-ascorbic acid (1000 g/ml) were 93.20 ± 1.3 % and 95.50 ± 2.08 %, respectively.

In vitro Anti-inflammatory Studies

Inhibition of Protein Denaturation

Table 6: Inhibition of protein denaturation Results

Values are represented as Mean ± SEM

Further investigation into the anti-inflammatory mechanism was performed by testing the EEFD's inhibitory impact on protein denaturation. It worked well to avert the heat-induced denaturation of albumin. At a dose 100 µg/ml, suppression was measured to be 73.1 ± 4.37%. At a concentration 100 µg/ml, the common anti-inflammatory medication diclofenac sodium inhibits at a maximum rate of 79.0 ± 0.84%. When compared to diclofenac sodium, for which the IC50 is 179.6 ± 7.32µg/ml, EEFD's result is lower, and at 98.05 ± 3.76µg/ml. Table 6 clearly illustrates this.

Proteinase inhibitory activity

Table 7: Proteinase inhibitory activity Results

Values represented as Mean ± SEM      

Table 14 shows that at several concentrations, EEFD has significant anti-proteinase action. At 100μg/ml, there was a maximum inhibition of EEFD of 80.3 ± 1.89 %. Diclofenac sodium showed 91.6 ± 0.95 % inhibition at a concentration of 100μg/ml. EEFD had an IC50 of 91.9 ± 4.12μg/ml, whereas diclofenac sodium had an IC50 of 88.74 ± 1.42μg/ml.

In vivo Anti-inflammatory Studies:

Table: Carrageenan-induced Paw edema in albino rats

Taking F. daldousiae orally showed promising anti-inflammatory effects. The 800 mg/kg dose of F. dalhousiae reduced edema volume significantly. An hour afterward (1.4±0.03ml). A significant edema decrease was seen after 3 hours (1.1± 0.05 ml). Edoema volume decreased similarly with 400 mg/kg (1.3± 0.04 to 0.7± 0.14 ml) dose. Comparing the results to common anti-inflammatory drugs such Diclofenac sodium (10mg/kg), considerable inhibition was shown after 5 hours (1.4 ± 0.03 to 0.4 ± 0.02 ml).

Biochemical parameters

Table 2: Complete Blood Picture Results

The CBP test shows an increase in the toxic and low dosage groups in the absolute number of WBC, Lym, Gran, Mid, and PLT as well as the percentage of Lym, Gran, Mid. HGB, HCT, MCV, MC, and MCHC levels are all within normal ranges, while there is a little rise in the RBC count as well. When comparing the high dosage to the standard, you'll notice a little increase in WBC.

C-reactive Protein Test

Table 3: C-reactive Protein Test Results