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Phytochemical Screening and Anti-inflammatory Properties of Henna Leaves (Lawsonia inermis)

In traditional West African setting, plants are commonly used for cosmetic and medicinal benefits such as antibacterial, antifungal, anti-inflammatory, antioxidant and antiparasitic effect of which Henna (Lawsonia inermis), a natural occurring plant of the lythraceae family is not an exception. A preliminary qualitative phytochemical screening and anti-inflammatory properties of Lawsonia inermis leaves extracted with N-butanol and ethyl acetate was investigated. Standard analytical procedures were observed, and statistical analysis using ANOVA version 20.0 was conducted to analyze for mean ± standard deviation (SD) and the least significant difference (LSD) obtained from the study. Results revealed the presence of flavonoids, tannins, terpenes, cyanogenic and cardiac glycosides; while In vivo anti-inflammatory activity wasobserved on a week old cockerel chicks induced carrageenan inflammation post extract administration, using aspirin tablet as control. The anti-inflammatory activity of the extract as portrayed could be attributed to the plants rich phytochemical potential. Original Research Article Chuku et al.; EJMP, 31(18): 23-28, 2020; Article no.EJMP.63255 24


INTRODUCTION
Henna or Hina (Lawsonia inermis), a flowering plant and lone specie of the Lawsonia genus. It is commonly used as a dye for various uses (skin, hair, finger nails and fabrics). The plant is localized in the Sahel, Central Africa and slightly in the Middle East. Aside its external use, it serves some medicinal and therapeutic purposes ranging from hepatoprotective and immunomodulatory effects, antimicrobial, antifungal and antioxidant properties with scientific evidence [1]. In south west Nigeria, the aqueous leaves of L. inermis is used as antimalaria decoction according to scientific research [1]. Reports by Aguwa [2] also captured the abortifacient activity of L. inermis methanol root extract in mice, albino rats and guinea pig in a dose dependent ratio.
Secondary metabolites produced by plants, which confers attributes like colour, flavour and natural defence to plants are referred to as phytochemicals [3]. Human health over time has been influenced positively by the contributory protective effect of phytochemicals against a variety of degenerative diseases [4,5]. A wide range of mechanism suggests that plant phytochemicals are important components of human and animal diets. These phytochemicals commonly obtained from vegetables, fruits and seeds act as antioxidants, modulators of gene expression and signal transduction pathways [5]; (Okwu, 2005). Irritants, cell damage and pathogens which are harmful to the body tissues constitute a complex biological response referred to as inflammation [6]. Molecular mediators, blood vessels and immune cells constitute a protective response leading to inflammation. It is essentially the elimination of initial causes of cell injury, necrotic cells and tissue damage, followed by the initiation of tissue repair (Tanko et al., 2008). The study evaluates the therapeutic efficacy of N-butanol and ethyl acetate extracts of L. inermis leaves on carrageenan induced inflammation in a week old cockerel chicks and to ascertain the respectively phytochemicals responsible for the anti-inflammatory activity.

Plant Collection and Identification
Fresh L. inermis leaves were harvested from farm stead in Elele, Ikwerre L.G.A., Rivers State. Harvesting was carried out during the day and in good condition. Plant identification was done by Mr. I. Ozioko (rtd), a renowned botanist and former staff of Botany Department, University of Nigeria, Nsukka, Enugu State.

Procurement of Chicks
Twenty white cockerels (Gallus gallus domesticus, strain shaver 5790) weighing between 40-55 g were procured from Jocarl Breeder Farm, Umudioga road, Elele, Rivers State, Nigeria, conveyed to the animal house of pharmacognosy, Madonna University, Nigeria. They were properly housed with soft wood shavings as beddings, vaccinated and had access to clean water and commercially available starter pellet diet (Top feed, Nigeria) ad libitum prior to the study, with a 12/12 hours light and dark cycle maintained.

Preparation of Plant Material
Harvested and identified fresh leaves of Henna (L. inermis) were properly washed with distilled water to get rid of debris, and then air dried in natural environment afterwards. The dried leaves were homogenized into fine powder and 250 g each of powdered leaves was poured into two 1000 ml round bottom flasks. A 1250 ml each of N-butanol and acetyl acetate was measured and poured into the two flasks respectively. The resultant mixture was allowed to stand for 24 hours with occasional shaking, prior filtration. The filtrate was evaporated, first with a porcelain bag, and then with Whatman No. 1 filter paper before a rotary evaporator was used to dry to paste with the aid of a thermostatic water bath at 40°C. The concentrate was transferred into storage amber bottles and then stored in the refrigerator at 4°C for use.

Preliminary Qualitative Phytochemical Screening
Quantitative phytochemical screening for the detection of secondary metabolites (phenols and flavonoids) was carried out on the plant extracts based on the methods of Harbourne, [7] and Evans [8].

Test for carbohydrates
Molisch's test: 0.5 g of the extract was boiled with 2ml of distilled water and filtered. To the filtrate, few drops of α-naphthol solution in ethanol (Molisch reagent) was added and concentrated sulphuric acid was then gently poured down the side of the tube to form a lower layer. A purple interfacial ring formed indicates the presence of carbohydrate.

Test for saponins
20 ml of distilled water was added to 0.5 g of the respective extracts and boiled on a hot water bath for 2 minutes and filtered. The filtrate was allowed to cool and then used for the following tests; 1. Frothing test: 5 ml of the filtrate was diluted with 15 ml of distilled water and shaken vigorously. The formation of a stable froth indicates the presence of saponins. 2. Emulsion test: To the frothing solution was added 2 drops of olive oil and the contents shaken vigorously. The formation of emulsion indicates the presence of saponins. 3. Haemolytic test: 2 ml of the plant extracts were respectively mixed with 2 ml of antiagglutinated blood (whole blood) and shaken. Haemolysis of the red blood cells indicates the presence of saponins.

Test for steroids
9 ml of ethanol was added to 1 g of the extract and refluxed for a few minutes and filtered. The filtrate was concentrated to 2.5 ml on a boiling water bath. 5 ml of hot distilled water was added to the concentrated solution, the mixture was allowed to stand for 1 hour and the waxy substance was filtered off. The filtrate was extracted with 2.5 ml of chloroform using separating funnel. 1 ml of absolute sulphuric acid was carefully added to 0.5 ml of chloroform extract to form a lower layer. A reddish brown interface indicates the presence of steroids.

Test for alkaloids
20 ml of 3% sulphuric acid in 50% ethanol was added to 2 g of the extract and heated on a boiling water bath for 10 minutes, cooled and filtered.

Test for glycosides
10 ml of 1% sulphuric acid was added to 0.5 g of the pastry extract in a test tube and boiled for 15 minutes on a water bath, then cooled and neutralized with 20% potassium hydroxide solution. 10 ml of a mixture of equal volume of fehling's solution I and II was added and boiled for 5 minutes. A brick red precipitate indicates the presence of glycoside.

Anti-inflammatory Assay
Carrageenan-induced paw edema is the most common method employed in the evaluation of anti-inflammatory therapies. The use of carrageenan stimulated inflammation and the determination of the anti-inflammatory effects of Lawsonia inermis leaves extract in 7 day old domestic chicks was experimented. The time course effect of foot withdrawal laterncy to a thermal stimulus and edema were examined over a 6 hour period following an intraplantar carragenan injection. The extract was suspended in sterile water by adding 2% of tween 20 to enhance the suspension of the extract. Varying concentrations (50, 100 and 200 mg/kg) of the extract suspensions were administered to the chicks (n=5) and this served as the negative control. Carrageenan (10 µl of 2% suspension in saline) was injected subplantar into the right footpad of the chicks. The foot volume was measured before injection and at hourly interval for 5 hours after injection using a digital venier calliper. The edema induced by the inflammation was quantified by measuring the difference in foot thickness before carrageenan injection and at the various time points. The values were individually normalized as a percentage change in foot edema relative to the corresponding values at time zero, and then averaged for the respective treatment groups. The percentage inhibition for each treatment was determined thus:

Data Analysis
Data obtained were statistically analyzed for mean ± standard deviation (SD), comparison of mean and LSD correlation was done using analysis of variance (ANOVA) version 20.0 and significance set at p<0.05.

RESULTS
The result of qualitative phytochemical screening of N-butanol and ethylacetate extracts of L. inermis leaves carried out in triplicate evaluations indicated significant (p<0.05) presence of beneficial phytochemicals with low levels of proteins and carbohydrates (Table 1).
Carrageenan induced edema has a course of action that lasts for approximately 2 hours post injection and the carrageenan induced inflammation was seen at all concentrations tested. The activities of the extract were compared with that of a reference standard nonsteroidal anti-inflammatory drug, diclofenac sodium which served as a positive control (see Tables 2 and 3).

Qualitative Phytochemical Screening of N-butanol and Ethylacetate Extracts of L. inermis Leaves
The qualitative phytochemical screening on Nbutanol and ethylacetate extracts of L. inermis leaves carried out in triplicate indicated the presence of flavonoids, alkaloids, glycosides, saponins, tannins and steroids, with flavonoids having the most significant level. On the contrary, there were absence of carbohydrate and protein.
Plants containing flavonoids and alkaloids have shown antispasmodic, anti-inflammatory and   antioxidative properties (Table 1). However, from the results, there was no difference in the extract type (N-butanol and actylacetate) used which was attributed to the similarity as regards polarity index.

Anti-inflammatory Activity of Nbutanol Extract of L. Inermis Leaves on Carrageenan-induced Edema in Chicks
The anti-inflammatory activity of N-butanol extract of L. inermis leaves on carrageenaninduced edema in chicks showed a significant (p<0.05) reduction with increase in time (30 to 240 mins) on the level of edema within each group when compared with IVP and VAI respectively. However, group 3 (50 mg/kg bwt) demonstrated the most effective anti-inflammatory and therapeutic efficacy followed by group 4 (100 mg/kg bwt). The observed effect and reduction in edema was time dependent (see Table 2).

Anti-inflammatory Activity of Ethylacetate Extract of L. inermis Leaves on Carrageenan-induced Edema in Chicks
The ethylacetate extract of L. inermis leaves on carrageenan-induced edema in chicks was observed with significant (p<0.05) difference in anti-inflammatory activity. The leave extract had a reducing effect on carrageenan-induced edema with group 3 (50 mg/kg btw) showing the most effective time dependent activity. The antiinflammatory activity was also observed to be consistent with increase in time ( Table 3).
The N-butanol and ethylacetate extracts of L. inermis leaves illustrated a mild antiinflammatory effect oncarrragenan-induced edema on chick paw which was compared statistically with diclofenac (standard drug). The observed effect was concentration and time dependent as observed across groups with a peak activity of 50mg/kg weight. Most human clinical studies on the safety and therapeutic activity of green lipped mussel extract (GMLE) was shown to useful(Ahmad et al.

CONCLUSION
The anti-inflammatory and therapeutic activity of L. inermis extracts (N-butanol and ethyl acetate) has been reported with little or no scientific evidence. This investigation demonstrates the possibility of a promising anti-inflammatory activity which is time and concentration dependent, with an optimum therapeutic range of 50 mg/kg body weight irrespective of the mode of extraction. This assertion on the antiinflammatory activity is affirmed by the result of the investigation.

CONSENT
It is not applicable.

ETHICAL APPROVAL
Strict compliance with National Institute of Health (NIH) Nigeria guideline for the care and use of laboratory animals was maintained and permission sought from the animal handling and ethics committee of Madonna University, Nigeria [MAU/EC/20/029].