ABSTRACT

Objectives:

In recent times, the use of natural remedies, which are rich in varieties of vitamins and flavonoids, for treatment of inflammation has increased substantially. These natural remedies are expected to be safe and economical when compared with other conventional allopathic drugs. Thus, existing research investigated the anti-inflammatory effect of Nelumbo nucifera fruit (NNF), in view of estimating its traditional and pharmacologic use against disorders associated with pain and inflammation.

Materials and Methods:

To estimate the antiinflammatory effect of NNF, carrageenan-induced paw edema method was employed with equally distributed (n=7) Wistar male rats (N=35). The paw edema was measured by volume displacement method with plethysmometer.

Results:

The NNF extract significantly reduced the inflammation of the paw and decreased the edema volume in rats administered carrageenan at all doses from the 3^rd to 5^th hour when compared to control, whose maximum percent reduction of edema was estimated as 100 mg/kg dose (that is, 73.92% at the 5^th hour after administration of carrageenan).

Conclusion:

NNF exhibited a strong antiinflammatory effect, due to its phytochemical constituents, including flavonoids, saponins, and tannins, all of which synergistically exert inhibitory effects on arachidonic acid metabolism, neutrophil degranulation, and enzyme systems that promote cell proliferation and regulation of complement system. However, more preclinical and clinical evaluations are mandatory to validate these findings.

INTRODUCTION

A lot of individuals who experience severe, inexorable, and excruciating pain resulting from any deleterious painful condition, such as myocardial infarction or injury secondary to accidents, rely primarily on opioids, in spite of their established adverse effects. Similarly, unceasing anti-nflammatory conditions, such as rheumatoid arthritis and osteoarthritis, are generally cured with non-steroidal antiinflammatory drugs (NSAIDs). Although NSAIDs are dominating in the market, the toxicity associated with their prolonged use cannot be overlooked. The most frequent toxicity associated with NSAIDs includes gastro-intestinal tract bleeding and ulcers.^1,2 Therefore, this instigates the need to develop new, safe, effective, economical, and innocuous analgesics.³

The use of herbal drugs is progressively becoming more popular, since they are natural and have no adverse effects.⁴ Basically, plant-derived drugs are taken randomly with no adequate knowledge of their local application for the treatment of various diseases. It is therefore important to appropriately guide the general population on the use of natural products. In addition, it is necessary to scientifically prove the effectiveness of these medicinal plants.⁵

Nelumbo nucifera (NNF) (Lotus), a member of the Nymphaeaceae family of plants, is commonly cultivated in hot and humid climate zones of Thailand, Pakistan, India, and China.⁶ Its fruit contains seeds and pods. The green colored pods offer add-on to the seeds, which are usually black in color, tough, and roundish in shape (diameter: 1.5x1.0 cm). They are organized in whorls.⁷ The seeds are the edible part of the fruit, which have to be skinned separately before consumption.⁸

The seeds of NNF are good sources of protein, fat, asparagines, unsaturated fatty acids, and starch. The key active components in the seeds are flavonoids, alkaloids, carbohydrates, gallic acid, and ample amount of various minerals, in addition to zinc, iron, potassium, sodium, and calcium.^9,10

A recently conducted study on NNF pods revealed the existence of numerous active bioactive principles, such as flavonoids, alkaloids, saponins, terpenoids, and tannins.¹¹ Procyanidin (flavonoid) was also extracted from NNF pods.⁶

Customarily, the fruits are used as a healthy component of Asian cuisine. They are also used as a traditional cure for various ailments, such as hypertension, palpitation, arrhythmia, fever, pain, inflammation, sleep disorders, chronic diarrhea, spermatorrhea, leucorrhoea, bad breath, leprosy, and menorrhagia.^12,13 Recent research revealed that the LD₅₀ value of NNF was higher than 5g/kg, whereas its neuropharmacological role was also established to be anxiolytic, antidepressant, and antiepileptic.^11,14 However, there insufficient information in literature regarding its ameliorative effect on inflammation. In this regard, this study aimed to evaluate the antiinflammatory activity of NNF, in view of justifying its traditional and pharmacologic use against disorders associated with pain and inflammation.

MATERIALS AND METHODS

RESULTS

DISCUSSION

Several individuals who experience severe, inexorable, and excruciating pain resulting from cancer, injury, or from various autoimmune disorders and other degenerative diseases rely on opioids, such as morphine, in spite of their established adverse effects. Although NSAIDs are dominating in the market, toxicity associated with their prolonged use cannot be overlooked.^1,2 This instigates the need to develop novel, safe, and effective substances for ameliorating the effect of inflammation.

Acute inflammation induced by carrageenan is the most appropriate technique for screening antiinflammatory agents. The time required for edema to appear in carrageenan-induced hind paw edema model in rats is often estimated by a biphasic curve. The initial phase of inflammation arises within an hour of carrageenan administration, due to trauma at the injection site, serotonin, and histamine element.²⁵ The 2^nd phase of edema starts from the 3^rd hour and is sensitive to cyclooxygenase (COX) inhibitors, such as NSAIDs.³

This study revealed the antiinflammatory effect of NNF, which incredibly decreased the paw edema volume at all doses from the 3^rd to 5^th hour as compared to control, with maximum percent reduction of edema estimated at 100 mg/kg (that is, 73.92% at the 5^th hour after administration of carrageenan). The first phase of the edema (that is, 1^st and 2^nd hour) was not affected by the fruit extract. Hence, in the present study, there was no inhibition of histamine and serotonin at the first phase of the test; however, COX pathway was effectively inhibited at the second phase of the test.

Flavonoids, which are also one of the significant constituents of NNF, exert its antiinflammatory effects via several mechanisms. One of these mechanisms pertains to their proposed capability of diminishing neutrophil degranulation. This represents the shortest possible approach to inhibit the liberation of arachidonic acid by neutrophils and other immune cells. Neutrophils carrying lipoxygenase produce chemotactic factors from arachidonic acid, which also stimulate the release of cytokines.^26,27

Certain flavonoids are capable of reducing complement system activation, thus diminishing the attachment of inflammatory cells to the endothelium, thus reducing the inflammatory response.²⁸

Saponins and tannins, which are also present in NNF, have also been reported to show inhibitory effects on arachidonic acid breakdown.²⁹ A recently conducted study has revealed the analgesic activity of NNF in various animal models, postulating that it may be connected with the synergistic actions of flavonoids, saponins, and tannins on arachidonic acid inhibition.³⁰ Therefore, it can be stated that the antiinflammatory effects of NNF are largely due to their flavonoids, saponins, and tannins content, which is believed to synergistically exert inhibitory effects on arachidonic acid metabolism, neutrophil degranulation, and enzyme systems that promote cell growth and regulates complement system.

CONCLUSION

The antiinflammatory activity of NNF demonstrated in this study was due to the existence of phytochemical constituents, which make it a useful agent for the treatment of patients with chronic inflammatory disorders. However, further preclinical and clinical evaluation is required to validate these findings.

Study design and methodology

This study was performed with the assistance of the laboratory services of the Department of Pharmacology, UoK, following approval from the Board of Advance Studies and Research, UoK.

Animals care

The research board, which comprise members of Faculty of Pharmacy, permitted the use of animals for the experiments in accordance with the protocols described by NIH and NACLAR.^15,16

Animals were kept in plastic cages and maintained at a temperature near 25°C and humidity of 50% to 60% in an interchanging twelve hour cycle. Each mouse was given free access to normal diet and water. Rats were carried to the workroom about an hour prior to the trials initiation. Prior to administration of the dose, complete health of the rats was assessed based on the absence of movements, edema, diarrhea, and ulceration during the acclimatization process using the laboratory settings for 7 days.

Preparation of extract

After obtaining fruits from the domestic fruit bazaar of Hyderabad, Pakistan in July 2015, they were initially presented to Pharmacognosy Department, UoK for identification and authentication and, afterward, the receipt no NNF-03 was assigned and deposited in the same department.

Crude extract was prepared through cold extraction procedure.^17,18 In brief, fruits (6 kg) were initially rinsed with clean water and seeds were manually separated from the fruit. Since the seeds have high contents of water, they need to be chopped first and left for 6 days to dry out in shade. The dried material obtained was thick and, as a result, the seeds need to be ground into fine powder. In contrast, pods were chopped once and allowed to dry in shade for 3 days. The dried pod material was in a coarse powder form. For better separation and collection of NNF constituents (secondary metabolites), they need to be chopped and dried separately before soaking them in ethanol (98%) for 30 days with occasional shaking.

Afterward, it was filtered with whatman no. 1 filter paper. Then, it was evaporated with a rotary evaporator under condensed pressure at a temperature of 40°C-45°C. The condensed material was freeze-dried with a freeze dryer (-30°C) and stored in a refrigerator. The final yield of the extract was 0.4 kg (dry weight).

Grounding of drugs

Carrageenan and gum tragacanth were obtained from Merck. Aspirin was purchased from a well-known pharmacy in Karachi.

2% tragacanth gum (powder form) was procured from Merck, which was used to prepare suspensions of 3 different dosages for the test group (NNF 50, 100, and 200 mg/kg). Control group was given tragacanth gum (10 mL/kg PO) as placebo. On each occasion, new suspensions were prepared for the dosage.^19,20

Carrageenan suspension (0.1 mL of 1% w/v) was prepared in normal saline and inoculated via the planter aponeurosis of right hind paw to induce hind paw edema in the experimental rats.²¹

Aspirin (300 mg tablet) was crushed and suspended in tragacanth gum (2%), which was then administered to the rats at the dosage of 150 mg/kg PO (as reference agent) using an orogastric tube.²²

Hind paw edema method

Hind paw edema method is an established technique for assessing the antiinflammatory effect of NNF extract, following the induction of edema in the hind paw of rat by carrageenan. Generally, carrageenan is used as a phlogistic agent, a substance that induces inflammation or edema.²³

The test was performed on 35 Wistar rats that were equally distributed into 5 groups (n=7). The control group was administered 2% tragacanth gum; the reference group was administered 150 mg/kg aspirin; and the 3 test groups were administered 50, 100, and 200 mg/kg NNF. All drugs were administered orally (PO) at an hour prior to the delivery of carrageenan injection. Carrageenan suspension (0.1 mL of 1% w/v) prepared in normal saline was introduced under the planter aponeurosis of right hind paw of rats to induce hind paw edema in the experimental rats.²¹

The paw edema was estimated by volume displacement technique using a plethysmometer (UGO Basile 7140, Italy), which, perhaps, is the most efficient method for estimating the antiinflammatory effect of a drug. Plethysmometer is a volume meter that is composed of water-filled Perspex cell, in which the hind paw of rat was submerged with the transducer, which records small changes in water level caused by volume displacement, as well as with a digital meter, which displays the exact volume of water being displaced by the edema in the hind paw of rats. The swelling was estimated as mL of edema at different time interval, that is, the paw volume before carrageenan administration (time=0 baseline) and paw volume after carrageenan administration from the 1^st to 5^th hour was noted. Changes in the paw volume estimated earlier, as well as the succeeding administration of phlogistic agent points toward the significance of edema. Lastly, percent reduction of paw swelling was estimated in terms of % inhibition as follows:²⁴

% inhibition: A-B/A x100

Where;

 A: The average paw volume for test groups

 B: The average paw volume for control group

Statistical analysis

The statistical analysis was done by applying Student’s independent samples t-test to the mean and standard error of mean. P values less than 0.05 were considered significant and p values less than 0.005 were considered highly significant. Analysis of data was performed by using IBM SPSS Statistics for Windows, Version 20.0 (IBM Corp., Armonk, N.Y., USA).

Hind paw edema method

Table 1 and 2 showed that the carrageenan injection produced a localized edema at the right hind paw of rats, which attained its peak at the 3^rd hour after the carrageenan administration, but gradually reduced after this period. Administration of 50, 100, and 200 mg/kg NNF significantly reduced the paw edema volume from the 3^rd to 5^th hour when compared to control, whose maximum percent reduction of edema was estimated as 100 mg/kg (that is, 73.92% at the 5^th hour after administration of carrageenan). The initial phase of the edema (1^st and 2^nd hour) was not affected by the fruit extract. On the other hand, 150 mg/kg aspirin substantially decreased paw edema volume from the 1^st to 5^th hour when compared to control. Maximum percent inhibition of edema (88.17%) was estimated at the 5^th hour after the carrageenan administration.