Evaluation of Phytochemical and in Vitro Anti-Inflammatory activity of Leaf and Fruit Extracts of Casuarina equisetifolia

Vani Mamillapalli¹*, Ratna Harika Chapala¹, Tejaswi Komal Sai Sareddu¹, Latha Sri Kondaveeti¹, Santhi Pattipati¹, Padmalatha Khantamneni²

¹Department of Pharmacognosy and Phytochemistry, Vijaya Institute of Pharmaceutical Sciences for Women, Enikepadu-521108, Vijayawada, Krishna (Dt.), Andhra Pradesh, India.

²Department of Pharmacology, Vijaya Institute of Pharmaceutical Sciences for Women, Enikepadu-521108, Vijayawada, Krishna (Dt.), Andhra Pradesh, India.

*Corresponding Author E-mail: vanimamillapalli@yahoo.co.in

ABSTRACT:

The plant Casuarina equisetifolia, commonly called as horse tail belonging to family Casuarinaceae is used traditionally for the treatment of infections, ulcers, cough, diarrhea etc. The plant is a rich source of tannins and flavonoids. In the current study the plant leaf, fruit aqueous and ethanolic extracts were determined for total flavonoid content followed by in vitro anti-inflammatory activity study by HRBC membrane stabilization and protein denaturation assays. The results indicate that highest amount of rutin equivalent flavonoids were present in ethanolic extract of leaf, aqueous and ethanolic extracts of fruit. Fruit extracts exhibited highest % inhibition of lysis of HRBC. Aqueous leaf and fruit extracts exhibited highest inhibition of protein denaturation, The results indicate that further in vivo studies, phytochemical isolation, characterization studies could be conducted for plant extracts

KEYWORDS: Casuarina equisetifolia, total flavonoid, anti-inflammatory, HRBC lysis, protein denaturation.

INTRODUCTION:

Medicinal plants are potential store houses of various secondary metabolites regarded as phytochemicals gifted to Mankind to lead a disease-free life. The currently used 74% of drugs were developed with the help of ethnobotanical information¹. Plant-based drugs have greater scientific and economic significance² with about 80% of the world’s inhabitants relying mainly on traditional medicines for their primary health care needs ³. The inflammatory response involves a complex array of enzyme activation, mediator release, cell migration, tissue breakdown and repair which are aimed at host defense and usually activated in most disease condition.

The practice of using plants, their parts or extracts as anti-inflammatory compounds is known since antiquity ⁴. Flavonoids, including around 6000 phenolic compounds, are products of the secondary metabolism of plants which can be a part of one’s diet via the consumption of many edible plants. Chemically, flavonoids have a polyphenolic structure that confers antioxidant activities on them⁵. Beyond antioxidant properties, some particular kinds of flavonoids have shown protective effects against inflammatory-mediated disorders such as cancer, cardiovascular diseases, gastrointestinal alterations and nervous system-related syndromes, such as depression, epilepsy, Alzheimer’s disease and neurodegenerative disease, insulin-resistance obesity among other pathologic conditions⁶. In an immunity, inflammation context, in inflammatory-mediated diseases, the six subclasses of the flavonoid compounds act by various mechanisms at molecular level, such as acting as antioxidants, modulating gene expression (i.e., cytokines, adhesion molecules) or enzyme activities, inflammatory response, mainly conducted by macrophages and neutrophils as an expression of the innate immune system activation; and the immune system concerning, above all, acquired immunity⁷. Currently much interest have been paid in the searching of medicinal plants with anti-inflammatory activity which may lead to the discovery of new therapeutic agent that is not only used to suppress the inflammation but also used in diverse disease conditions where the inflammation response is amplifying the disease process⁸.

Casuarina is a genus of 17 species in the family Casuarinaceae, native to Australasia. It is common along the coast on beaches, rocky coasts, hill side and open forest in both wet and dry zones. It is also cultivated as ornamental plant for wind-breaks, or as a medicinal plant in some tropical countries in South pacific^9,10,11. It is an ornamental plant grown in Pakistan, India, tropical Africa and Sri Lanka¹². The plant is traditionally used for the treatment of constipation, cough, dysentery, gonorrhea, nervous disorders, acne, throat infections and stomach ulcer^13,14,15. Seeds were used as anthelmintic, antispasmodic and antidiabetic¹⁶. The plant has been reported for various activities suchas a polyherbal gel containing plant extract has shown anti-acne activity in rats¹⁷. Various parts of the plant have been reported to posess analgesic¹⁸, hyperglycemic^19,20, antihyperlipidemic¹⁰, antioxidant²¹, anti-inflammatory activity of bark, root extracts^21,22, antibacterial, cytotoxic²³, spasmolytic²⁴, hepatoprotective¹⁹, and nephroprotective²⁵ properties. The plant contains maximum amount of phenolics, tannins, flavonoids and terpenoids²². Casuarine is the alkaloid reported from this plant¹¹. With this view the present research was focussed to carry out the preliminary phytochemical screening, total flavonoid content and in vitro anti-inflammatory studies by RBC membrane stabilization, and protein denaturation assay methods on the leaf and fruit extracts of the plant Casuarina equisetifolia belonging to family Casuarinaceae.

MATERIALS AND METHODS:

The plant materials, leaves and fruits of Casuarina equisetifolia were collected in the month of July during afternoon from the grounds of Vijaya Institute of Pharmaceutical Sciences for Women, Enikepadu, co-ordinates 16°32′45″N 80°34′12″E of Vijayawada rural region. The plant sample was identified from the plant taxonomist Dr. P. Satya Narayana Raju, department of Botany and Microbiology, Acharya Nagarjuna University, Guntur. The photographs of the collected plant materials were depicted in figure 1, figure 2, figure 3, and figure 4 respectively. The leaves, and fruits were powdered coarsely after cleaning and dryng. They were extracted by soxhlation using water and ethanol. The collected extracts were dried under vacuum and preserved in refrigerator for future use.

Fig. 1: Casuarina equisetifolia plant

Fig. 2: Fresh fruiting branches of C. equisetifolia

Fig. 3: Dried fruiting branches of C. equisetifolia

Fig. 4: Fresh and dried fruits of C. equisetifolia

Preliminary phytochemical screening:

The plant aqueous and ethanolic extracts were subjected to preliminary phytochemical screening using standard methods^26,27,28 (Table 1).

Quantative determination of flavonoids:

The extract (1.5ml) of different concentrations (0.2, 0.4, 0.6, 0.8, and 1mcg/ml) was added to 4 ml distilled water and 0.3ml 5% NaNO₂ (Finar) solution, incubated (Bio-tech) for 5 minutes. 0.3ml 10% AlCl₃(Finar), was added and allowed to stand for 6 minutes. 2ml of 1M NaOH (Finar) was added and finally the volume was made up to 10ml with distilled water. It was allowed to stand for 15 minutes. The absorbance was read at 510nm using UV-Visible spectrophotometer. (Lab India). The assay was carried out in triplicate^29,30. The results were calculated by the given formula, and expressed as rutin (Finar) equivalent in mg/g dry extract, were given in table 2, figure 5.

C = c.V/m

Where,

C – Total phenolic compounds mg/g of plant extract

c – The concentration of standard established from the calibration curve mg/ml

V – The volume of extract in ml

m - The weight of pure plant extract

In-vitro anti-inflammatory activity by RBC membrane stabilization assay:

The blood was collected from healthy human volunteers who have not taken any NSAIDS for 2 weeks prior to the experiment and mixed with equal volume of Alsever’s solution (2% dextrose, 0.8% sodium citrate, 0.5% citric acid and 0.42% NaCl) (Merck) and centrifuged (Lab India) at 3,000rpm. The packed cells were washed with isosaline and a 10% suspension was made. Various concentrations of extracts were prepared (0.1, 0.3, and 0.5mcg/ml) using distilled water and to each concentration 1ml of phosphate buffer, 2ml hyposaline and 0.5ml of HRBC suspension were added. It was incubated (Biotech) at 37⁰C for 30 min and centrifuged at 3,000rpm for 20 min. The haemoglobin content of the supernatant solution was estimated spectrophotometrically (Lab India) at 296nm. Diclofenac Sodium (Merck) was used as reference standard and a control was prepared omitting the extracts ³¹. The percentage inhibition of hypotonicity induced haemolysis was calculated using the formula

Where, OD= Optical density or absorbance

In-vitro anti-inflammatory activity by protein denaturation assay:

The ability of plant extracts was studied using inhibition of albumin denaturation technique. The reaction mixture consisted of different concentrations (0.1, 0.3, and 0.5 mcg/ml) of plant extracts and 1% aqueous bovine albumin fraction. The reaction mixture was incubated at 37^oC for 10 min and then heated at 51^oC for 20 min. The turbidity was measured at 237nm using UV-Visible spectrophotometer after cooling and was compared with the reference compound diclofenac Sodium³². Experiments were performed in triplicate. The percentage inhibition of hypotonicity induced haemolysis was calculated using the above formula.

RESULTS AND DISCUSSION:

The preliminary qualitative screening revealed the presence of alkaloids, glycosides, tannins, flavonoids, terpenoids, phytosterols and saponins in aqueous and ethanolic extracts of leaf and fruit parts of the plant (Table 1). The rutin equivalent flavonoid content was found to be more in leaf ethanolic extract (ELCE) (0.009±0.015mg RE/gm), fruit aqueous (AQFCE) (0.009±0.0017mg RE/gm) and ethanolic (EFCE) (0.009±0.001mg RE/gm) extracts of C. equisetifolia (Table 2; figure 5). It was found to be less in leaf aqueous extract (AQLCE) (0.003±0.035mg RE/gm) when compared to other extracts of the plant.

Table 1: Qualitative preliminary phytochemical screening of aqueous and ethanolic leaf and fruit extracts of C.equisetfolia

S. No.	Phytochemical	AQLCE	ELCE	AQFCE	EFCE
1.	Alkaloids	+	+	+	+
2.	Glycosides	+	+	+	+
3.	Tannins or phenolics	+	+	+	+
4.	Flavonoids	+	+	+	+
5.	Triterpenes	+	+	+	+
6.	Steroids	+	+	+	+
7.	Saponins	+	+	+	+

AQLCE- Aqueous extract of leaves of C.equisetfolia, ELCE-Ethanolic extract of leaves of C.equisetfolia, AQFCE- Aqueous extract of fruits of C.equisetfolia, EFCE- Ethnolic extract of fruits of C.equisetfolia

Table 2: Quantitative Phytochemical screening of flavonoids of aqueous, ethanolic leaf and fruit extracts of C.equisetfolia

S. No.	Phytochemical (mg RE/gm)	AQLCE	ELCE	AQFCE	EFCE
1.	Flavonoids	0.003±0.035	0.009±0.015	0.009±0.0017	0.009±0.001

Values represented mean±S.D. of three parallel measurements. AQLCE- Aqueous extract of leaves of Casuarina equisetfolia, ELCE-Ethanolic extract of leaves of C.equisetfolia, AQFCE- Aqueous extract of fruits of C.equisetfolia, EFCE- Ethnolic extract of fruits of C.equisetfolia, RE-Rutin equivalent.

Fig. 5: Standard calibration of rutin for flavonoids

Exposure of Red Blood Cells (RBC) to injurious substances such as hypotonic medium, heat, chemicals such as or phenylhydrazine, or methyl salicylate results in the lysis of the membranes, accompanied by haemolysis and oxidation of haemoglobin ³³. Since Human Red Blood Cell (HRBC) membranes are similar to lysosomal membrane components ³⁴, the inhibition of hypotonicity and red blood cell membrane lysis was taken as a measure of the mechanism of anti-inflammatory activity. AQFCE (97.3±0.0007*) and EFCE (97.3±0.0005*) exhibited highest inhibition of haemolysis at 0.5 µg/ml concentration than all other groups at p<0.05*level of significance. The % inhibition was better than standard group diclofenac (60.1±.0.0005*). The results were expressed in table 3 and figure 6. The results of the study were further supported by the anti-inflammatory studies of bark and root extracts of plant ^{21, 22}. The results of RBC membrane stabilization method are in line with the findings of Puspal et al., 2017 ³⁵. The high total rutin equivalent flavonoid content of fruit aqueous and ethanolic extracts indicates that flavonoid components may be responsible for in –vitro RBC membrane stabilization.

Table 3: % Inhibition of haemolysis by HRBC membrane stabilization assay

S. No.	Groups	% Inhibition of HRBC haemolysis
S. No.	Groups	0.1 µg/ml	0.3 µg/ml	0.5 µg/ml
1	STD, Diclofenac	48.0±0.0005*	50.1±0.0011	60.1±.0.0005*
2	AQLCE	25.3±0.0077*	25.8±.0.0027*	73.1±0.0005
3	ELCE	30.0±0.0004	50.0±0.0005	73.0±0.0005
4	AQFCE	40.0±0.048*	49.7±0.0005*	97.3±0.0007*
5	EFCE	46.7±0.0142	53.0±0.0005	97.3±0.0005*

Values represented mean ± S.D., n=3, at p<0.05^* by one-way ANOVA, Dunnett’s test. AQLCE- Aqueous extract of leaves of C.equisetfolia, ELCE-Ethanolic extract of leaves of C.equisetfolia, AQFCE- Aqueous extract of fruits of C.equisetfolia, EFCE- Ethanolic extract of fruits of C.equisetfolia,

Fig. 6: Inhibition of haemolysis by HRBC membrane stabilization assay

Inflammation has been implicated in the pathogenesis of many diseases including arthritis, stroke, and cancer³⁶. Protein denaturation has been well correlated with the occurrence of the inflammatory response and leads to various inflammatory diseases including arthritis³². According to Opie³⁷, tissue injury during life might refer to denaturation of the protein constituents of cells or of intercellular substance. Hence, the ability of a substance to inhibit the denaturation of protein signifies apparent potential for anti-inflammatory activity.

Table 4: % Inhibition of protein denaturation

S. No.	Groups	% Inhibition of protein denaturation
S. No.	Groups	0.1 µg/ml	0.3 µg/ml	0.5 µg/ml
1	STD, Diclofenac	20.3±0.0097	53.9±0.0097*	65.2±0.0097*
2	AQLCE	67.6±0.0215*	85.9±0.0313*	92.4±0.0034*
3	ELCE	59.9±0.0013	76.1±0.0026*	91.9±0.0012
4	AQFCE	64.7±0.0208*	70.7±0.0252	78.4±0.0274*
5	EFCE	75.3±0.0172*	77.0±0.0302*	77.6±0.0165*

Values represented mean ± S.D., n=3, at p<0.05^* by one-way ANOVA, Dunnett’s test. The values were compared to standard. AQLCE- Aqueous extract of leaves of C.equisetfolia, ELCE-Ethanolic extract of leaves of C.equisetfolia, AQFCE- Aqueous extract of fruits of C.equisetfolia, EFCE- Ethnolic extract of fruits of C.equisetfolia

Fig. 7: % Inhibition of protein denaturation

AQLCE (92.4±0.0034*) and ELCE (91.9±0.0012) showed highest % inhibition of protein denaturation at 0.5µg/ml. The % inhibition of protein denaturation by leaf aqueous and alcoholic extracts was better than standard drug diclofenac (Table 4; figure 7). The results of the study are in line with the findings of Nurul et al., 2016³⁸. A number of flavonoids are reported to possess anti-inflammatory activity in vitro and in vivo. Although not fully understood, several mechanisms of action are proposed to explain in vivo anti-inflammatory action. The important mechanism for anti-inflammatory activity is inhibition of eicosanoid generating enzymes including phospholipase A2, cyclooxygenases and lipoxygenases, thereby reducing the concentrations of prostanoids and leukotrienes^39,40. The total rutin equivalent flavonoid content of aqueous and ethanolic leaf extracts (AQLCE and ELCE) was found to be 0.003±0.035 and 0.009±0.015mg RE/gm. Rutin was found to possess anti-inflammatory effects against pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs) induced by HMGB1 and the associated signaling pathways⁴¹. Rutin was found to be more effective in chronic inflammation⁴². In the present protein denaturation study AQLCE and ELCE exhibited highest percentage inhibition of protein denaturation which might be due to total flavonoid content.

CONCLUSION:

Medicinal plants are natural store house of remedies since ages. The results of the present study indicate that the C.equisetifolia plant extracts could be effective agents for the treatment of various inflammatory disorders. The study also supported the ethno medicinal claims of the plant C.equisetifolia. Hence, further in vitro studies can be carried out to prove the efficacy of plant extracts.

ACKNOWLEDGEMENT:

The authors are thankful to Vijaya Institute of Pharmaceutical Sciences for Women, Enikepadu, Vijayawada, Krishna (Dt.), A.P., India, for their kind encouragement and support.

CONFLICT OF INTEREST:

The authors declared no conflict of interest.

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Received on 20.04.2020 Modified on 23.05.2020

Asian J. Pharm. Tech. 2020; 10(3):143-148.

DOI: 10.5958/2231-5713.2020.00025.2