ANTI-INFLAMMATORY ACTIVITY POTENTIAL OF ARECA CATECHU LEAF EXTRACTS-AN IN-VITRO STUDY

ANTI-INFLAMMATORY ACTIVITY POTENTIAL OF ARECA CATECHU LEAF EXTRACTS-AN IN-VITRO STUDY

G. D’ Souza, J. Fernandes * and S. D’ Souza

Department of Pharmaceutical Chemistry, NGSM Institute of Pharmaceutical Sciences, Nitte (Deemed to be University), Paneer, Deralakatte, Mangalore, Karnataka, India.

ABSTRACT: Areca catechu L. (Arecaceae) is a species of palm mainly grown in Asian countries for seed crop. Areca nut leaves are commonly used in traditional systems of medicine against various ailments. There is a need for identifying native natural plant sources to acquire their recognized medicinal properties, which may widen them to use as new therapeutics for various diseases. Phytochemical investigation of the leaves of Areca catechu L. yielded a alkaloid- Arecoline, from the chloroform extract; a flavonoid- Apigenin, from the methanol extract and a steroid- Stigmasterol, from petroleum ether extract. In-vitro methods were used to evaluate the pharmacological activity of the leaves of Areca catechu L. Anti-inflammatory activity of the leaves of Areca catechu was estimated by using standard methods like protein denaturation method using bovine serum albumin fraction and egg albumin and by HRBC membrane stabilization method. The result suggests that the leaves possess good antioxidant as well as anti-inflammatory activity.

Keywords: Areca catechu, Antioxidant, Anti-inflammatory activity

INTRODUCTION: Herbal medicine, also known as phytotherapy, is the practice of treating different illnesses with natural remedies. This involves all facets of herbal medicine, i.e. plants with strong acts for people with gentle actions ¹. Herbal medicines is the key of traditional medicine systems like Ayurveda, Sidha and Unani ². Traditional medicine, especially herbal medicine, is seen as a major provider of healthcare around the world, particularly in rural and remote areas ³.

For the treatment of different health problems, medicinal plants appear to be an interesting source of natural products ^{4, 5}. Even with the advances of the modern medicines, in many developing countries herbal medicines play a major role in the treatment of many diseases. In India around 85% of the population still uses the crude plant preparations for treating various types of ailments and diseases. The plant material which is used as the API in the herbal formulations is readily available in and around the rural areas which has made herbal medicines cheaper compared to the modern medicines ⁶.

Inflammation is the process of defence triggered by living tissue during an illness or injury. This initiates phase of healing by removing the injurious agents and around tissue debris ⁷.

Anti-inflammatory drugs are classified as agents that either prevent or decrease inflammation that can occur as a result of interactions between physical damage, infection, heat and antigen-antibody ⁸.

Steroidal and non-steroidal anti-inflammatory medications that, when used as long-term therapies, may cause significant side effects, such as gastrointestinal and peptic ulcer bleeding ⁹. An alternative source of new compounds with anti-inflammatory activity is natural products ¹⁰. Medicinal plants play an important role in the development of new and potent drugs due to the availability of secondary metabolites with clinical curative effects ¹¹. Areca catechu L. (Family: Arecaceae) commonly known as Areca palm or Betel nut palm or Betel palm. These are widely cultivated and grown more or less throughout the year in India and many other Asian countries ¹². The name betel nut palm or betel palm is because, its fruits are often chewed with the betel leaf and lime ¹³. The fruits of the palm are traditionally used in diarrhoea, urinary disorders, digestive aid, expelling intestinal worms, stimulant effect and breathe freshening effect ¹⁴. While the nuts of the tree have been reported for many activities there is no literature available for the isolation of phytoconstituents of the leaves of the plant. To confirm the active constituents of the leaves, the present study is designed. With this background the leaves of the plant are selected for screening of anti-inflammatory activity ¹⁵.

MATERIALS AND METHODS:

Plant Collection: The leaves of Areca catechu required for the study was collected from in and around Mangalore, Dakshina Kannada (dist), Karnataka; in the month of June and July 2017. The leaves were authenticated by Dr. K. V. Nagalakshamma, Head of the Dept. of Botany, St. Aloysius College, Mangalore. A voucher specimen (No.16PC005) is deposited in NGSM Institute of Pharmaceutical Sciences, Paneer, Mangalore.

Extraction: The leaves of Areca catechu L. were cleaned and shade dried for about 7 days. The shade dried leaves were then ground into coarse powder using a mechanical grinder. The coarse powder of leaves of Areca catechu L. were subjected to cold maceration extraction using chloroform and methanol as the solvents ¹⁶. Cold maceration was done in two parts of the plant powder (100 g) for all the solvents, each for 7 days. After the extraction, the macerated powder was filtered using a muslin cloth. The marc obtained was air-dried. The filtrate obtained was subjected to steam distillation, to concentrate the extract and the solvent was recollected and was used for further extraction process. A dark green residue was obtained on further concentrating and evaporating the extract on a water bath. The dried extract thus obtained was kept in the desiccator and was used for further phytochemical and pharmacological investigation.

Preliminary Phytochemical screening Qualitative Analysis: The chloroform, methanol extracts of leaves of Areca catechu L. was subjected to preliminary qualitative phytochemical screening, to determine the different chemical groups present in it. Chemical tests were carried out as per the standard methods ¹⁷.

Screening of Anti-Inflammatory Activity By In-vitro Methods: Denaturation of tissue proteins and lysis of RBC membrane are the prominent causes of inflammatory diseases. During inflammation, there are lyses of lysosomes which release their component enzymes that produce a variety of disorders. Non-steroidal anti-inflammatory drugs (NSAIDs) exert their beneficial effects by either inhibiting the release of lysosomal enzymes or by stabilizing the lysosomal membranes ¹⁸.

These drugs in spite of their potency in relieving pain are also associated with some serious side effects on prolonged duration of usage which include gastric bleeding, bone marrow disturbance, ulceration, liver and kidney dysfunction ¹⁹. However, plant-derived drugs are used to treat most of the inflammatory diseases even today because of their least or no side effects ²⁰.

Protein Denaturation by Bovine Serum Albumin Method: The reaction mixture consisting of test extracts and 1% aqueous solution of bovine albumin fraction was prepared. The pH of the reaction mixture was adjusted using a small amount of 0.1N HCl. Sample mixture consists of 0.45 ml of bovine serum albumin solution and 0.05 ml of the extract solution ²¹.

0.5 ml of this sample mixture was taken in test tubes and were incubated at 37 °C for 20 min and then to the above sample mixture 2.5 ml of phosphate buffer saline was added.

The absorbance of the sample mixture was taken at 660 nm.  Percentage inhibition of protein denaturation was calculated using the following formula.

% inhibition = (Abs_control – Abs_test/ Abs_control) × 100

Where Abs_control is the absorbance of the control and Abs_test is the absorbance in the presence of test extracts

Protein Denaturation by Egg Albumin Method: The reaction mixture consisting of 0.45 ml of 5% egg albumin solution and 0.05 ml of the test extract solution was taken in a test tube and these tubes were incubated at 37 °C for 20 min.

After incubation to the above solution 2.5 ml of the phosphate buffer saline was added. The absorbance of the reaction mixture was taken at 660 nm. Percentage inhibition of protein denaturation was calculated using the following formula ²¹.

% inhibition = (Abs_control – Abs_test/ Abs_control) × 100

Where Abs_control is the absorbance of the control and Abs_test is the absorbance in the presence of test extracts.

Human Red Blood Cell Membrane Stabilization Method:

Collection of blood sample: The human red blood cells required for the experiment was collected from a healthy volunteer who had not consumed any anti-inflammatory drugs (NSAID’s) for 2 weeks prior to the experiment ^{22, 23}.

Preparation of Human Red Blood Cell (HRBCS) Suspension: The fresh whole human blood (10 ml) collected was transferred to the centrifuge tubes with equi volume of Alsever’s solution. The tubes were centrifuged at 3000 rpm for 10 min and the packed cells were washed thrice with equal volume of iso-saline solution. The volume of the cells was measured and a 10% v/v suspension was reconstituted using iso-saline solution ²⁴.

Heat-Induced Haemolysis: 1 ml of the test extract each of concentration ranging from 50-300 µg/ml, was taken in a test tube, to this 1 ml of freshly prepared phosphate buffer was added along with 2 ml of saline solution. Finally, 0.5 ml of freshly prepared HRBC suspension was added. Diclofenac sodium was used as the standard drug and in the control tube saline was taken omitting the test sample. All the test tubes were incubated at 37 °C for 30 min. After incubation, the contents were then centrifuged at 3000 rpm for 20 min. The supernatant solution was collected and was estimated spectrophotometrically at 560 nm. The percentage inhibition of lysis was calculated using the following formula.

% inhibition = (Abs_control – Abs_test/ Abs_control) × 100

Where Abs_control is the absorbance of the control and Abs_test is the absorbance in the presence of test extracts

Results:

Preliminary Phytochemical Investigation: Preliminary phytochemical screening of both the chloroform extract and methanol extract of the leaves of Areca catechu revealed the presence of alkaloids, carbohydrates, flavonoids, triterpenoids, resins, steroids and tannins.

In-vitro Anti-Inflammatory Activity:

Bovine Serum Albumin Denaturation Method:

TABLE 1: EFFECT OF CHLOROFORM AND METHANOLIC EXTRACT OF ARECA CATECHU ON BOVINE SERUM ALBUMIN DENATURATION

All values are expressed in terms of ± SEM and are found to be significant when compared to control P<0.05

FIG. 1: BSA DENATURATION METHOD

Egg Albumin Denaturation Method:

TABLE 2: EFFECT OF THE CHLOROFORM AND METHANOLIC EXTRACT OF ARECA CATECHU ON EGG ALBUMIN DENATURATION

All values are expressed in terms of ± SEM and are found to be significant when compared to control P<0.05

HRBC Membrane Stabilization Method:

TABLE 3: EFFECT OF CHLOROFORM AND METHANOLIC EXTRACT OF ARECA CATECHU ON HRBC MEMBRANE STABILIZATION TEST

All values are expressed in terms of ± SEM and are found to be significant when compared to the control P<0.05.

FIG. 2: EGG ALBUMIN DENATURATION METHOD         

FIG. 3: HRBC MEMBRANE STABILIZATION METHOD

Discussion:

In-vitro Anti-Inflammatory Studies

Bovine Serum Albumin Denaturation Assay: The inhibition of denaturation of bovine serum albumin by heat was studied with chloroform as well as methanolic extract of the leaves of Areca catechu as the test sample and diclofenac sodium as the standard drug.

The extracts showed concentration dependant increase in the percentage inhibition values as similar to that of the standard drug. The IC₅₀ value of chloroform extract was found to be 131.95 and that of methanolic extract was 125.60, whereas the standard value was 110.19. The absorbance values decreased as the concentration increased, which was recorded at 660 nm, showed significant anti-inflammatory activity.

Egg Albumin Denaturation Assay: In this method, the only difference when compared to bovine serum albumin denaturation assay is that the bovine serum albumin fraction was replaced with egg albumin. The percentage inhibition of leaves extract of Areca catechu increased with increasing concentration. The IC₅₀ values of chloroform extract were found to be 159.06, and that of methanolic extract was found to be 146.36, whereas the standard value was 137.61. The decrease in the absorbance value with the increasing concentration taken at 660 nm shows significant anti-inflammatory activity.

HRBC Membrane Stabilization Method: The chloroform and methanolic extract of leaves of Areca catechu showed concentration dependant increase in percentage inhibition with HRBC membrane stabilization method. The IC₅₀ values of chloroform extract were found to be 185.97, and that of methanolic extract was found to be 167.87, whereas the standard value was 152.64. The decrease in the absorbance value with an increase in concentration suggests that the anti-inflammatory activity is observed in this study.

CONCLUSION: Preliminary phytochemical screening of chloroform and methanolic extracts of leaves of Areca catechu gave positive results for the presence of alkaloids, reducing sugars, flavonoids, triterpenoids, glycosides, resins, steroids, and tannins; and gave negative results for saponins and proteins. Anti-inflammatory activity of the leaves of Areca catechu was estimated by using standard methods like protein denaturation method using bovine serum albumin fraction and egg albumin; and by HRBC membrane stabilization method.

The chloroform extract, as well as the methanolic extract of the Areca catechu, showed significant percentage inhibition when compared to the control. Diclofenac sodium was used as the standard drug. The in-vitro anti-inflammatory activity showed positive results, thus promoting their use in the management of inflammation. From the studies carried out on leaves of Areca catechu L., it is evident that it has significant anti-inflammatory activity. The side effects caused by the currently available drugs like NSAIDs and opioid analgesics used in managing pain and inflammation made to search for newer agents with less toxicity, more effective, and more economical, thus promoting their use in the traditional system of medicine.

The results obtained in the present study are encouraging and offer the scope of further study. In any case where a group of plant part which are reported to be used in the treatment of any specific disease contain relatively higher levels of a specific element in comparison with other samples. These propositions offer further studies on these plants, constituents concerned and the corresponding diseases. Suitable animal experiments could be designed to further investigate the efficacy of these plants in treating the diseases and also the remedial roles of these elements.

ACKNOWLEDGEMENT: The authors are grateful to NGSM Institute of Pharmaceutical Sciences and Nitte (Deemed to be University) for providing the necessary facilities to carry out the research work.

CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

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    How to cite this article:

    D’ Souza NG, Fernandes J and D’ Souza S: Anti-inflammatory activity potential of Areca catechu leaf extracts-an in-vitro study. Int J Pharm Sci & Res 2021; 12(9): 5065-71. doi: 10.13040/IJPSR.0975-8232.12(9).5065-71.

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