EVALUATION OF ANTI-INFLAMMATORY ACTIVITY AND DETERMINATION OF TOTAL FLAVONOIDS AND TANNIN CONTENTS OF LAGENARIA SICERARIA ROOT

EVALUATION OF ANTI-INFLAMMATORY ACTIVITY AND DETERMINATION OF TOTAL FLAVONOIDS AND TANNIN CONTENTS OF LAGENARIA SICERARIA ROOT

Hemayet Hossain*¹, A.F.M Shahid-Ud-Daula ², Ismet Ara Jahan¹, Ishrat Nimmi, K. M. Rahat Maruf ² and Md. Musfizur Hassan ³

BCSIR Laboratories, Dhaka, Bangladesh Council of Scientific and Industrial Research (BCSIR) ¹, Dr. Qudrat-E-Khuda Road, Dhaka-1205, Bangladesh

Pharmacy Department, Noakhali Science and Technology University ², Noakhali-3814, Bangladesh

Department of Pharmacy, Jahangirnagar University ³, Savar, Dhaka-1342, Bangladesh

ABSTRACT:

The crude ethanolic extract of the root of Lagenaria siceraria (Molina) Standley (Family: Cucurbitaceae), was evaluated for its possible anti-inflammatory activity and determination of total flavonoids and tannin contents growing in Bangladesh. The anti-inflammatory activity was studied using carrageenan and histamine-induced rat paw edema test at different doses (200 and 400 mg/kg body weight) of the ethanol extract. At the dose of 400 mg/kg body weight, the extract showed a significant anti-inflammatory activity both in the carrageenan and histamine-induced oedema test models in rats showing 59.57% and 58.86% reduction in the paw volume comparable (P<0.01) to that produced by the standard drug indomethacin (63.12% and 66.45%) at 4h respectively. The percentage inhibition of the oedema paw volume by the 400 mg/kg body weight of the extract was also statistically significant (P<0.05; P<0.01) compared favorably with the indomethacin treated animals at 1, 2 and 3 h in both models. The total flavonoids and tannins content were calculated as quite high in ethanolic extract (244.29 mg/g of quercetin equivalent and 84.59 mg of tannic acid equivalent respectively). Acute toxicity test showed that the plant might be safe for pharmacological uses. Therefore, the obtained results tend to suggest the acute anti-inflammatory activity as well as total flavonoids and tannin contents from the ethanolic extract of the root of Lagenaria siceraria and thus provide the scientific basis for the traditional uses of this plant part as a remedy for pain and inflammations.

Total tannins

INTRODUCTION: Lagenaria siceraria (Molina) Standley (Family: Cucurbitaceae), commonly known as Bottle guard, an excellent fruit in the nature having composition of all the essential constituents that are required for good health of humans ¹. It is also good source of minerals and amino acids.

The fruit of Lagenaria siceraria contains minerals, amino acids, triterepeniode (cucurbitacins B, D, G, H), two sterols (fucosterols, campesterol), ascorbic acid ^{2, 3}. The different extracts of L. siceraria fruit have been reported to possess diuretic, hepatoprotective, cardioprotective, antihyperlipidemic and immuno-modulatory effects ^{4, 5, 6}.

Acute and chronic inflammatory diseases remain one of the world’s major health problems ^{7, 8}. Inflammation is the response of living tissues to injury. It involves a complex array of enzyme activation, mediator release, extravasations of fluid, cell migration, tissue breakdown and repair ^{9, 10}.

Non-steroidal anti-inflammatory drugs (NSAID) are among the most commonly prescribed drugs due to their consistent effectiveness in the treatment of pain, fever, inflammation and rheumatic disorders. However, their use is associated with adverse effects at the level of digestive tract, ranging from dyspeptic symptoms, gastrointestinal erosions and peptic ulcers to more serious complications, such as over bleeding or perforation ¹¹.

Therefore to overcome the toxicity of NSAID, the development of new anti-inflammatory drugs is still necessary and the natural product such as medicinal plants could lead in discovering new anti-inflammatory drugs with less undesirable effects ¹². Now-a-days attention is being focused on the investigation of the efficacy of plant-based drugs used in the traditional medicine because they are cheap, have little side effects and according to WHO, about 80% of the world population still rely mainly on herbal remedies ¹³.

Since no literature is currently available to substantiate anti-inflammatory activity as well as flavonoids and tannin contents from the ethanolic extract of the root of Lagenaria siceraria growing in Bangladesh, the present study was designed to provide scientific evidence for its use as a traditional folk remedyby investigating the anti-inflammatory activity as well as total flavonoids and tannins content from the root of L. siceraria that also confirm its use as a remedy for pain and inflammations.

MATERIALS AND METHODS:

Collection and identification of Plant Materials: Theplant (roots) L. siceraria was collected at December, 2010 from Noakhali, northeast district of Bangladesh and was identified by Bangladesh National Herbarium, Mirpur, Dhaka (Accession number-DACB-39286).

Preparation of Ethanolic Extract: The roots of L. siceraria were freed from any of the foreign materials. Then the roots were chopped and air-dried under shed temperature followed by drying in an electric oven at 40^oC. The dried plant materials were then ground into powder. About 500g of powdered material was taken in a clean, flat-bottomed glass container and soaked in 1.4 liters of 80% ethanol. The container with its contents was sealed and kept for a period of 4 days accompanying occasional shaking and stirring. The ethanolic extract was filtered by Buchner funnel and the filtrate was concentrated with rotary evaporator at bath temperature not exceeding 40^oC to have gummy concentrate of extract (yield approx. 13.26%).

Test for different Chemical Groups: The crude ethanolic extract was tested for its different chemical groups as alkaloids, flavonoids, gums, reducing sugars, saponins, steroids and tannins ¹⁶. In each test 10% (w/v) solution of the extract in ethanol was taken.

Experimental animals and Drug: For the screening of in vivo anti-inflammatory activity male rats of Wister strain weighing 175-202 g were used. The animals were housed under standard Laboratory (at Pharmacology Laboratory of BCSIR, Chittagong) conditions maintained at 25±1°C and under 12/12 h light/dark cycle and feed with Balanced Trusty Chunts and water ad libitum. All experimental protocols were in compliance with BCSIR Ethics Committee on Research in Animals as well as internationally accepted principles for laboratory animal use and care.

The standard drug Indomethacin was used for this study and purchased from Square Pharmaceuticals Ltd, Bangladesh.

Chemicals: Tannic acid, quercetin, carrageenan, folin-ciocalteu phenol reagent and histamine were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Tween 80, aluminium chloride, potassium acetate and sodium carbonate were of analytical grade and purchased from Merck (Darmstat, Germany).

Acute Toxicity Test: The acute toxicity of L. siceraria ethanolic extract was determined in rats according to the method of Hilaly et al ¹⁵ with slight modifications. Rats fasted for 16h were randomly divided into groups of five rats per group. Graded doses of the extract (200, 400, 800, 1600 and 3200 mg/kg p.o.) were separately administered to the rats in each of the groups by means of bulbed steel needle. All the animals were then allowed free access to food and water and observed over a period of 48h for signs of acute toxicity. The number of deaths within this period was recorded.

Anti-inflammatory Activity:

1. Carrageenan-induced oedema test: Carrageenan induced rat hind paw edema was used as the animal model of acute inflammation according to the method of Lanhers et al ¹⁶. In this experiment, the rats were divided into four groups of five animals each. Group I (control) received 2% Tween 80 in normal saline (2 ml/kg). Group II (Positive control) received 10 mg/kg body wt. of Indomethacin orally. Group III and IV received 200 and 400 mg/kg body wt. of the extract orally respectively. Acute inflammation was induced in all the four groups by sub plantar injection of 0.05 ml of its suspension of Carrageenan with 2% Tween 80 in normal saline in the right Paw of the rats 30 minutes after the oral administration of the tested materials. The paw volume was measured with a micrometer screw gause at 1, 2, 3 and 4h after the administration of the drug and the extract. The percentage inhibition of inflammatory effect of the extract was calculated using the following expression:

Percentage inhibition of inflammation

= [(V_c-V_t)/V_c] × 100

Where V_c is the average degree of inflammation by the control group and V_t is the average degree of inflammation by the test group (Table 2).

2. Histamine-induced Oedema Test: Using the method of Perianayagam et al ¹⁷, the paw oedema was produced by sub-plantar administration of 0.1% freshly prepared solution of histamine into the right hind paw of the rats. In this experiment, twenty rats were divided into four groups of five animals each. Group I (control) received 2% Tween 80 in normal saline (2 ml/kg). Group II (Positive control) received 10 mg/kg body wt. of Indomethacin orally. Group III and IV received 200 and 400 mg/kg body wt. of the extract orally respectively. Acute inflammation was induced in all the four groups by sub plantar injection of 0.1 ml of Histamine with 2% Tween 80 in normal saline in the right hind paw of the rats 1h after the oral administration of the tested materials. The paw volume was measured with a micrometer screw gause at 1, 2, 3 and 4h after the administration of the drug and the extract. The percentage inhibition of inflammatory effect of the extract was calculated using the same formula for carrageenan-induced paw oedema.
3. Determination of Total Flavonoids Content: Aluminium chloride colorimetric method was used for determination of total flavonoids concentration of the ethanol extract¹⁸. The extract (0.5 ml, 1:10 g ml^-1) in ethanol were separately mixed with 1.5 ml of methanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water. It was allowed to stand for 30 min at room temperature and the absorbance of the reaction mixture was measured at 415 nm with a double beam Analykjena UV/Visible spectrophotometer (Model 205, Jena, Germany). Total flavonoids content was determined as mg of Quercetin equivalent per gram using the equation obtained from Quercetin calibration curve y= 4.7385x + 0.0355; R² = 0.9993.
4. Determination of Total Tannins Content: The tannins were determined using the Folin-ciocalteu Phenol reagent as reported by Amorim et al ¹⁹. Briefly, 0.1 ml of the sample extract was added with 7.5 ml of distilled water and added 0.5 ml of Folin-ciocalteu Phenol reagent, 1 ml of 35% sodium carbonate solution and dilute to 10 ml with distilled water. The mixture was shaken well, kept at room temperature for 30 min and absorbance was measured at 725 nm. Blank was prepared with water instead of the sample. A set of standard solutions of tannic acid is read against a blank. The results of tannins were expressed in terms of tannic acid in mg/g of dry extract. Total tannin content was determined as mg of tannic acid equivalent per gram  using the equation obtained from a standard tannic acid calibration curve y=4.5692x-0.2538, R²=0.9953.

Statistical Analysis: Data were presented as mean ± Standard deviation (S.D). Statistical analysis for animal experiment was carried out using one-way ANOVA followed by Dunnet’s multiple comparisons using SPSS Data Editor for Windows, Version 11.5.0 (SPSS Inc., U.S.A.). The results obtained were compared with the control group. p values < 0.05 were considered to be statistically significant (p denotes probability).

RESULTS:

Phytochemical Screening: Results of different chemical tests on the ethanolic extract ofL. siceraria root showed the presence of saponin, gums, steroid, tannins and significantly presence of flavonoids (Table 1).

TABLE 1: Results of different group tests of ethanolic extract of L. siceraria root.

+: Positive result; - : Negative result; ++: significantly positive

Acute Toxicity Test: In acute toxicity study, oral administration of graded doses (200, 400, 800, 1600 and 3200 mg/kg p.o.) of the ethanol extract of L. siceraria to rats did not produce any significant changes in behaviour, breathing, cutaneous effects, sensory nervous system responses or gastrointestinal effects during the observation period. No mortality or any toxic reaction was recorded in any group after 48h of administering the extract to the animals. L. siceraria was safe upto a dose level of 3200 mg/kg of body weight.

Anti-inflammatory Activity:

1. Carrageenan-induced Paw Oedema: The anti inflammation effect of the ethanolic extract of the root of L. siceraria using carrageenan induced oedema tests is expressed in Table 2. In this test, the positive control (Indomethacin) significantly (p<0.05; p<0.01) decreased the paw edema at 1h to 4h after carrageenan injection compared to saline with inhibition 56.86% to 63.12%. A maximum oedema paw volume of 1.41±0.05 mm was observed in the control rats, 4 h after the carrageenan injection. Rats with the extract at 400 mg/kg body weight significantly decreased (p<0.05; p<0.01) the carrageenan-induced oedema paw volume from 1h to 4h compared to the standard drug indomethacin at a dose of 10 mg/kg body weight. The inhibition percentage of the oedema paw volume by the 400 mg/kg body weight of the extract was also found statistically significant when it was compared with the indomethacin treated animals at 1, 2, 3 and 4 h. The highest reduction in the paw volume by the 400 mg/kg body weight was 59.57% was comparable to that of the indomethacin (63.12%) at 4 h.

TABLE 2: EFFECT OF ETHANOL EXTRACT OF L. SICERARIA ROOT AND INDOMETHACIN ON CARRAGEENAN-INDUCED OEDEMA PAW VOLUME IN MALE WISTAR RATS

Values in brackets denote percentage inhibition of the oedema paw volume. Values are expressed as mean±SD; Values are calculated as compared to control using one way-ANOVA followed by Dunnet’s Test; * indicates P < 0.05; ** indicates P < 0.01 vs. control; n = 5.

Histamine-induced Paw Oedema: Table 3 showed the anti-inflammation effect of the ethanolic extract ofL. siceraria root using histamine-induced paw oedema tests. In the histamine-induced oedema test, a maximum oedema paw volume of 1.58 ± 0.09 mm was observed in the control rats, 4 h after the histamine injection. Rats pre-treated with the extract at 400 mg/kg body weight significantly decreased (p<0.05; p<0.01) the histamine-induced oedema paw volume from 1h to 4 h compared to the standard drug indomethacin at a dose of 10 mg/kg body weight. The percentage inhibition of the oedema paw volume by the 400 mg/kg body weight of the extract was also statistically significant (p<0.05; p<0.01) compared favorably with the indomethacin treated animals at 1, 2, 3 and 4 h. The maximum reduction in the paw volume by the 400 mg/kg body weight was 58.86% compared to the indomethacin (66.45%) at 4 h.

TABLE 3: EFFECT OF ETHANOL EXTRACT OF L. SICERARIA ROOT AND INDOMETHACIN (STANDARD DRUG) ON HISTAMINE-INDUCED OEDEMA PAW VOLUME IN MALE WISTAR RATS

Values in brackets denote percentage inhibition of the oedema paw volume. Values are expressed as mean±SD; Values are calculated as compared to control using one way-ANOVA followed by Dunnet’s Test;  * indicates P < 0.05; ** indicates P < 0.01 vs. control; n = 5.

Total flavonoids content: The total flavonoids content was calculated as significant in ethanolic extract of L. siceraria 244.29 mg/g of quercetin equivalent per gm of dry extract (Table 4).

TABLE 4: Total flavonoids content of ethanol extract of L. siceraria root.

The values are expressed as mean ± standard deviation (n=3).

Total Tannin Content: The total tannin content was calculated as quite high in ethanolic extract 84.59 mg/g of tannic acid equivalent (Table 5).

TABLE 5: TOTAL TANNIN CONTENT OF ETHANOL EXTRACT OF L. SICERARIA ROOT.

The values are expressed as mean ± standard deviation (n=3).

DISCUSSION: The anti-inflammatory activity was studied using two established method namely carrageenan and histamine-induced rat paw edema test at different doses (200 and 400 mg/kg body weight) of the ethanol extract of L. siceraria root.

Carrageenan-induced oedema involves the synthesis or release of mediators at the injured site. These mediators include prostaglandins, especially the E series, histamine, bradykinins, leucotrienes and serotonin, all of which also cause pain and fever ²⁰. Inhibitions of these mediators from reaching the injured site or from bringing out their pharmacological effects normally ameliorate the inflammation and other symptoms. Development of oedema induced by carrageenan is commonly correlated with early exudative stage of inflammation ²¹. Carrageenan oedema is a multimediated phenomenon that liberates diversity of mediators.

It is believed to be biphasic; the first phase (1h) involves the release of serotonin and histamine while the second phase (over 1h) is mediated by prostaglandins, the cyclooxygenase products, and the continuity between the two phases is provided by kinins ¹⁷. Since carrageenan-induced inflammation model is a significant predictive test for anti-inflammatory agents acting by the mediators of acute inflammation ^{22, 23}, the results of this study are an indication that L. siceraria can be effective in acute inflammatory disorders.

The extract also exhibited pronounced reduction in the oedema produced by histamine. This result tends to suggest that the anti-inflammatory activity of the extract is possibly backed by its anti-histamine activity. The antihistaminic effect of the extract increased with increase in the dose of the extract. Histamine is an important inflammation mediator, potent vasodilator substance and also increases the vascular permeability ^{24, 25, 26}.

Since the extract effectively suppressed the oedema produced by histamine, it showed that the extract exhibited anti-inflammatory actions by inhibiting the synthesis, release or action of inflammatory mediators such as histamine, serotonin and prostaglandins. . This study has shown that the ethanol extract of the root of L. siceraria possessed a significant anti-oedematogenic effect (P<0.01) on paw oedema induced by carrageenan and histamine compared favorably with the standard drug (indomethacin) in treated rats.

The anti-inflammatory activity of the ethanolic extract of L. siceraria may also be proved due to the presence of flavonoids in a significant amount (244.29 mg quercetin equivalent per g of dry extract). Flavonoids (or bioflavonoids) are naturally occurring compounds, containing in vascular plants. These compounds have been considered to possess anti-inflammatory properties, both in vitro and in vivo ²⁷. Numerous studies have proposed that flavonoids act through a variety mechanisms to prevent and attenuate inflammatory responses and serve as possible cardioprotective, neuroprotective and chemo-preventive agents ²⁸.

Phytochemically, the roots of L. siceraria have been also reported to yield tannins. The total tannin amount was calculated as quite high in ethanolic crude extract (84.59 mg/g of tannic acid equivalent) (table 5). Tannins are important compounds known to be potent cyclooxygenase-1 inhibitors and with anti-phlogistic activity ²⁹. The mechanisms of anti-inflammatory activity may be related to the anti-phlogistic action of the tannins.

Non-steroidal anti-inflammatory drugs (NSAID) such as indomethacin used in this study are known to inhibit cyclooxygenase enzymes I and II which are implicated in the production of inflammation-mediating agent prostaglandin E₂ (PGE₂) from arachidonic acid ^30,31. Therefore, the pattern of anti-inflammatory activity exhibited by this extract was similar to that of indomethacin.

CONCLUSION: Since the plant extract reduced significantly the formation of oedema induced by carrageenan and histamine, the root of L. siceraria exhibited acute anti-inflammatory activity. The potential of the extract of L. siceraria as acute anti-inflammatory agent may be due to the presence of phytoconstituents like flavonoids, tannins, phenolics ⁴ and might be responsible for its activity. Again, no mortality was recorded in the acute toxicity test; it showed that the plant might be safe for use.

Therefore, it can be revealed that the ethanolic extract of L. siceraria rootpossess acute anti-inflammatory activity and justify its use as a traditional folk remedy forinflammation, pain etc. However, a more extensive study is necessary to determine the exact mechanism(s) of action of the extract and its active compound(s).

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    Hossain H, Shahid-Ud-Daula AFM, Jahan IR, Nimmi I, Maruf KMR and Hassan MM: Evaluation of Anti-Inflammatory activity and determination of Total Flavonoids and Tannin contents of Lagenaria siceraria Root. Int J Pharm Sci Res 2012; Vol. 3(8): 2679-2685.

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