SCREENING OF ANALGESIC AND ANTI-INFLAMMATORY ACTIVITY OF GLORIOSA SUPERBA IN RODENTS

SCREENING OF ANALGESIC AND ANTI-INFLAMMATORY ACTIVITY OF GLORIOSA SUPERBA IN RODENTS

B. Srinivasa, Basavaraj C. Kotinatot, Aruna Bushan and P. Madhav *

Department of Pharmacology, Apollo Institute of Medical Sciences and Research, Chittoor, Andhra Pradesh, India.

ABSTRACT: Background: Traditionally Gloriosa superba is being used as a medicinal plant in outskirts of belagavi, we evaluated and compared the analgesic and anti-inflammatory activities of hydroalcoholic extract of dried aerial parts (rhizomes) of G. superba with standard drugs. Methods: This experimental animal study was carried out in the Research Laboratory, Department of Pharmacology, BIMS, Belagavi. Prior permission from institutional ethical committee and institutional animal ethics committee was taken. Albino rats and Albino mice were used. Acute toxicity study was also done according to OECD guideline no. 425 and test doses were decided accordingly. The experimental models of Eddy’s hot plate method was used to study the analgesic activity whereas formalin induced peritonitis and cotton pellet granuloma models were used for anti-inflammatory action. Statistical analysis was performed using Mann-whitney U test and chi-square test (SPSS software version 22). Results: In eddy’s hot plate method, for G. superba extract of 400mg/kg, percentage increase in reaction time of at 120 mins was 60.49%, in formalin induces peritonitis method, for G. superba extract of 400mg/kg, percentage inhibition of peritoneal exudate was 47.14%. In cotton pellet induced granuloma method, for G. superba extract of 400mg/kg, percentage inhibition of granuloma formation was 50.32%. All the results of 4 different groups in each method showed significant difference in their respective pharmacological activities (Chi Square values: 14.4, 14.4, 75.2). Conclusion: Hydroalcoholic extract of G. superba has shown significant analgesic and anti-inflammatory activity in our study.

Keywords: Analgesic activity, Anti-inflammatory activity, Gloriosa superba

INTRODUCTION: Pain is an unpleasant sensation and emotional experience associated with actual or potential tissue damage or described in terms of such damage ¹. The pathophysiology of pain involves 2 components, peripheral nociception and central mechanism.

There are 2 main classes of pain - Integumental pain and visceral pain ².   Treatment available to reduce the pain includes NSAIDS, opioids, anticonvulsants and muscle relaxants and these drugs also have adverse effects which are common nowadays.

Inflammation is a normal protective response to tissue injury caused by physical trauma, noxious chemicals or microbiologic agents ³. Drug therapy of inflammation has always been debatable. The drugs most commonly used now in the treatment of inflammation are glucocorticoids and non-steroidal Anti-inflammatory drugs (NSAIDS). Most of these drugs are known to cause more or less deleterious side effects even leading to hospital admissions due to ADRs. Hence there is an always scope for continuous research to identify more effective and safer agents in the therapy of inflammation which are potent and nontoxic. Indian traditional system of medicine uses herbs that have anti-inflammatory property. Gloriosa superb is one of many medicinal plants extensively used in southern parts of India for treatment of diverse conditions predominantly pertaining to pain and inflammation. Gloriosa superb is a good abortifacient ⁴. Its seeds and tubers are used mainly for treating gout and rheumatism ⁵. Roots are purgative, cholagogue, antihelminthic, astringent and germicidal. It also cures leprosy, swelling, piles, chronic ulcers and colic pain in bladder. Powder is used for treatment of rheumatic fever. Various parts of the plant are used in sores, tumours and syphillis. Extract of plant is also used as CNS depressant ⁶.   Paste is an antidote in snake bite ⁷.

Scientific basis for traditional use of this plant is not much explored. Hence, the study is chosen to screen the analgesic and anti-inflammatory activity of hydro alcoholic extract of rhizome of Gloriosa superb through analgesic model, acute and subacute inflammatory models and to provide pharmacological basis for its use in traditional medicine for various analgesic and inflammatory disorders.

FIG. 1A:

MATERIALS AND METHODS: Our study was carried out in the Research Laboratory, Department of Pharmacology, Belagavi Institute of Medical Sciences, Belagavi. Ethical clearance was taken before commencing the study and CPCSEA number stated below.

Collection of Plant Product: Rhizomes of G. superba were collected from the local ayurvedic pharmacy of Belagavi, Karnataka, India and authenticated by an authorized person in botany. The dried parts of rhizomes were powdered and stored in an air tight container.

Preparation of Extract: The powder (2kg) was soaked in 50% ethanol and 50% distilled water (i.e. hydroalcoholic extract) at 50-55°C for 7 days. After 7days, solvent from the total extract was distilled off and the concentrate was evaporated on a water bath to a syrupy consistency and then evaporated to dryness ⁸.

Selection of Animals: Wistar albino rats of either sex weighing between 150 and 200 g and albino mice of either sex weighing between 20 and 30 g were obtained from BIMS animal house registered under the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and maintained under good laboratory conditions. These animals were used for the acute toxicity, anti-inflammatory and analgesic activities. The study was approved by Institutional Ethics Committee for animal experimentation (Approval no: BIMS/IAEC/PG/06/2018 Dated- 13/11/2018).

The animals were stabilized for 1 week, maintained under standard laboratory conditions and  were given standard pellet diet and water ad libitum throughout the course of the study. The animals were handled gently to avoid undue stress, which could result in an increased adrenal output.

Acute Toxicity Studies: Toxicity study for hydroalcoholic extract of G. superba was carried out as per OECD guideline number 425 ⁹.  Healthy adult albino wistar rats (200-250 g) were used. Animals were fasted overnight prior to dosing. The body weight of each animal was determined to calculate the dose. Hydroalcoholic extract of G. superba was administered in the dose of 2000 mg/kg body weight orally to one animal which survived. Thereafter, four other animals were dosed sequentially. All the animals were closely observed for 14 days. As no fatality was observed, LD₅₀ was estimated to be greater than 2000 mg/kg. After performing a pilot study, we decided to use 200 mg/kg and 400mg/kg of hydroalcoholic extract G. superba for all the experiments.

Screening Methods for Analgesic Activity:

Eddy’s Hot Plate Method: Mice of either sex were weighed and divided into 4 different groups (n = 6 in each group). Group I served as control. Group II (Pentazocine 46.8 mg/kg body weight) served as standard and groups III and IV were treated with extracts at a dose of 200 and 400 mg/kg body weight, respectively. The reaction time of animals was noted down on hot plate at 0, 30, 60, 90, 120 and 150 minutes after the treatment. The basal reaction was the time taken by observing hind paw licking or jump response (whichever appeared first) in animals while placed on hot plate, which was maintained at constant temperature 55° C. A cut off period of 10 seconds was observed to avoid damage to the paws. The percentage increase or decrease in reaction time as index of analgesia at each time interval was calculated ¹⁰.

Percentage increase in reaction time = {(R_t / R_c) – 1} x 100

Where R_t is reaction time in treated group and R_c is reaction time in control group.

Screening Methods for Anti-Inflammatory Activity: Rats were randomly divided into four groups of 6 each. Group I served as control. Group II (diclofenac 13.5 mg/kg body weight) served as standard and groups III and IV were treated with G. superba extracts at a dose of 200 and 400 mg/kg body weight, respectively. Each rat was fed with respective drug one hour prior to the administration of phlogestic agent.

Formalin Induced Peritonitis in Rats: The method described by Teotino et al ¹¹ was used to study the acute inflammatory reaction induced by intraperitoneal injection of formalin in rats. Under aseptic precautions, 1ml of 1% formalin was injected intraperitoneally in each rat. At the end of four hours, peritoneal exudates was collected by opening the ventral abdominal wall. Volume of the peritoneal exudates in diclofenac (13.5mg/kg) and G. superba treated group was compared with the control group.

Percentage inhibition of peritoneal exudate formation in diclofenac and G. superba treated group was determined by the following formula.

Percentage inhibition of peritoneal exudates = {1-(V _t/ V_c)} х100

Where, V_c- Mean volume of exudates in control group.

V_t- Mean volume of exudates in treated test groups.

Cotton Pellet Induced Granuloma in Rats: The method described by Meier et al ¹² was used to study the chronic inflammatory reaction induced by subcutaneous cotton pellet implantation. Sterile cotton pellets weighing 20 mg each was implanted subcutaneously in the axillary region of both forefeet and groin under anaesthesia ¹³.

Each rat was orally administered with respective drug once daily for 7 consecutive days.

On 8^th day, the animals were sacrificed, cotton pellet with granulation tissue was removed, dried at 60°C in hot air oven for 24 hours and dry weight was determined.

Percentage inhibition of granuloma formation in diclofenac (13.5mg/kg) and Gloriosa superba treated rats was determined by the following formula:

Percentage inhibition of granuloma formation = {1-(T_t / T_c)} х 100

Where, T_c- Weight of granuloma tissue in control group

T_t- Weight of granuloma tissue in treated test groups

RESULTS: Results were analyzed using Mann-whitney U test and chi-square test (SPSS software version 22). Table 1 and Figure 1 indicate that hydroalcoholic extract of rhizomes of Gloriosa superba in dose of 200mg/kg and 400mg/kg shows significant analgesic activity with percentage increase in reaction time of 47.84% & 60.84% respectively in eddy’s hot plate method. Further, above two test doses of 200mg/kg and 400mg/kg of Gloriosa superba when compared with standard drug – Pentazocine (79.78%), showed no significant difference in analgesic activity (P=0.18 - P ˃ 0.05, P = 0.748 - P ˃ 0.05). Thus, test drug activity is not comparable with the standard drug pentazocine (P ˃ 0.05). Analgesic activity of two test doses of test drug (200mg/kg and 400mg/kg) was not comparable (P = 0.407 - P ˃ 0.05).

All the results of four groups showed significant difference in analgesic activity (Chi square = 14.412).

Table 2 and Figure 2 indicate that hydroalcoholic extract of rhizomes of Gloriosa superba in dose of 200mg/kg and 400mg/kg shows significant anti-inflammatory activity with percentage inhibition in peritoneal exudates of 40.35% & 47.14% respectively in acute model of inflammation. Further, above two test doses of 200mg/kg and 400mg/kg of Gloriosa superba when compared with standard drug Diclofenac (49.28%), showed no significant difference in anti-inflammatory activity (P=0.145 - P ˃ 0.05, P = 0.63 - P ˃ 0.05). Thus, test drug activity is not comparable with the standard drug diclofenac (P ˃ 0.05).

Anti-inflammatory activity of two test doses of test drug (200mg/kg and 400mg/kg) was not comparable (P = 0.256 - P ˃ 0.05). All the results of four groups showed significant difference in anti-inflammatory activity (Chi square = 14.441). Table 3 and Fig. 3 indicate that hydroalcoholic extract of rhizomes of Gloriosa superba in dose of 200mg/kg and 400mg/kg shows significant anti-inflammatory activity with percentage inhibition in granuloma formation of 35.54% & 50.32% respectively in sub-acute model of inflammation. Further, above two test doses of 200mg/kg and 400mg/kg of G. superba when compared with standard drug – Diclofenac (55.98%), showed significant difference in anti-inflammatory activity (P=0.001 - P ˂ 0.05, P = 0.004 - P ˂ 0.05). Thus, test drug activity is comparable with the standard drug diclofenac (P ˂ 0.05). Anti-inflammatory activity of two test doses of test drug (200mg/kg and 400mg/kg) was comparable or statistically significant (P = 0.001 - P ˂ 0.05). All the results of four groups showed significant difference in anti-inflammatory activity (Chi square = 75.174).

TABLE 1: ANALGESIC EFFECT OF VARIOUS DRUGS ON MICE USING EDDY'S HOT PLATE METHOD

TABLE 2: ANTI-INFLAMMATORY EFFECT OF VARIOUS DRUGS ON RATS USING FORMALIN INDUCED PERITONITIS METHOD

TABLE 3: ANTI-INFLAMMATORY EFFECT OF VARIOUS DRUGS ON RATS USING COTTON PELLET INDUCED GRANULOMA METHOD

TABLE 4: REPORTED ADDITIONAL SPECIAL PHARMACOLOGICAL PROPERTIES OF DIFFERENT PARTS OF GLORIOSA SUPERBA WITH DIFFERENT EXTRACTS

FIG. 1: SHOWING PERCENTAGE (%) INCREASE IN REACTION TIME IN EDDY’S HOT PLATE MODEL

FIG. 2: SHOWING PERCENTAGE (%) INHIBITION OF MEAN VOLUME OF PERITONEAL EXUDATES IN FORMALIN INDUCED PERITONITIS MODEL

FIG. 3: SHOWING PERCENTAGE (%) INHIBITION OF GRANULOMA FORMATION IN COTTON PELLET INDUCED GRANULOMA MODEL

DISCUSSION: In the present era there are number of different inflammatory disorders for which there are variety of anti-inflammatory drugs but associated with side effects like gastric ulcers, GI bleeding, renal damage etc. So, anti-inflammatory drugs with minimal side effects to be explored therapeutically in huge magnitudes for variety of inflammatory disorders and used accordingly.

In this context, it is exciting to note the reports of medicinal values of several indigenous plant preparations. Uses of these preparations are also consistently highlighted in ayurveda but without proper scientific knowledge. Hence, some of different extracts of different plants have been tested for pharmacological activities.

Globally there is extensive traditional use of a plant by name “Gloriosa superba” as there is significant therapeutically potential like analgesic, anti-rheumatic, anti-pyretic activities etc. Thereby we tried to evaluated the analgesic and anti-inflammatory activities of this plant by specific models respectively as described above to support and provide pharmacological basis in future for the use in our allopathy practice.

In Eddy’s hot plate model the extract of G. superba increased reaction time from 40.84% in test dose 200mg/kg to 60.49% in 400mg/kg to thermal pain, which simulates central antinociceptive test ¹⁴ and thereby standard drug pentazocine was used to compare and simulate central antinociception with test drug.  Inhibition of histamine or kinin pathway may reduce pain. The results of the present study also showed that extract exhibited a comparable magnitude of antinociceptive activity in both models of pain which suggested that the phytochemical constituents are responsible for the analgesic effect. The analgesic activity of some flavonoids ¹⁵ and terpenoids already has been reported suggesting that these or similar constituents may be responsible for the analgesic effect of the extract. The results of the present study indicated that the hydroalcoholic extract of G. superba might contain constituents capable of relieving or modifying responses to pain caused by either thermal or chemical stimulation of the noiceptors mediated by both central and peripheral mechanisms. Both the two doses of extract exhibited maximum percentage protection due to increase in reaction time (6.30 ± 0.19, 7.16 ± 0.39) at 90 minutes after drug administration thereby clearly indicating good analgesic activity of Gloriosa superba. The results obtained from this study is similar to study done by John et al ¹⁶ and support our study.

Formalin induced peritonitis model effectively demonstrates the use of formalin to induce peritonitis in rats by injecting intraperitoneally and results showed significant effect of standard anti-inflammatory drug diclofenac and Gloriosa superba as there was significant percentage protection or percentage inhibition in the volume of peritoneal exudates (40.34%. with G S 200mg/kg and 47.14%. with G S 400mg/kg) when compared to control thereby clearly indicating anti-inflammatory activity of Gloriosa superba.

Formalin was choosen as the preferred phlogestic agent to induce acute inflammation because it adequately resembles the chain of events underlying inflammation in patients. Other phlogestic agents which can be used to induce acute inflammation are carrageenan (sulphated polysaccharide obtained from seaweed Rhodophyceae), lipopolysaccharide, zymosan based on varying degree and duration of action needed. As there are no similar studies done of this model for this plant G. superba, comparison cannot be done.

Cotton pellet induced granuloma model effectively demonstrates and measures both exudative and proliferative phases of inflammation. 200mg/kg of G. superba extract showed statistically significant decrease in dry granuloma weight (35.54%, P=0.001 hence P ˂ 0.05) when compared to control group. This shows that hydroalocoholic extract of this plant is effective against subacute and chronic inflammation but its activity was inferior in the dose of 200mg/kg when compared to standard drug diclofenac. 400mg/kg dose of this plant extract showed statistically significant decrease in dry granuloma weight (50.32%, P=0.001 hence P ˂ 0.05) when compared to control group. The activity of G. Superba in 400mg/kg was almost similar to standard drug diclofenac. The results obtained from this model for G. superba is slightly superior to study done by John, et al ¹⁶ and support the study. As there are very few studies done previously of this model for this plant, our results cannot be compared with other studies. Because medicinal plants are rich in flavonoids and every group of flavonoids has a capacity to act as antioxidants ¹⁷, different studies of various parts of this  plant has been demonstrated the scientific basis for analgesic and anti-inflammatory activity by isolation of various chemical constituents of this plant. Among them, flavones and catechin components acting as most powerful flavonoids for protecting the body against ROS ¹⁸.

The other flavonoid components such as quercetin, kaempferol, myricetin and rutin have antioxidant, anti-inflammatory, antiviral and antiallergic, as well as anticancer activities ^{19, 20},  G. superba tubers contain colchicines, benzoic and salicylic acid, sterols and resinous substances like as colchicines, 3-demethyl colchicine, 1,2-didemethyl colchicine, 2, 3-didemethyl colchicine, N-formyl, N-deacetyl colchicines, colchicocide, gloriosine, tannins and superbine ²¹.

Colchicine is the major compound isolated from the seed and rhizome of this plant ²² and other important compound is gloriosine ^{23, 24}, In addition, G. superba tubers hold 0.25% colchicine apart from containing sitosterol, glucoside, β-and gamma lumicolichicines, β-sitosterol, flucoside and 2-H-6-MeO benzoic acid and flowers contain luteolin and N-formylde-Me-Colchicine ^{25, 26}.  Apart from these important phytochemical constituents of G. superba and their respective pharmacological properties, there are more additional special properties of this plant with different extracts which are extensively explored and documented by various reports of different studies (As mentioned in Table 4. In our study we conclude scientifically that rhizomes of Gloriosa superba showed good analgesic & anti-inflammatory activity in mice & rats.

ACKNOWLEDGEMENT: We thank statistician and director of our institution for their contribution in statistics of our study & granting permission to conduct the study in the institution respectively.

CONFLICTS OF INTEREST: NIL

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    How to cite this article:

    Srinivasa B, Kotinatot BC, Bushan A and Madhav P: “Screening of analgesic and anti-inflammatory activity of Gloriosa superba in rodents”. Int J Pharm Sci & Res 2023; 14(12): 6000-07. doi: 10.13040/IJPSR.0975-8232.14(12).6000-07.

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