EVALUATION OF ANTI ULCER ACTIVITY OF HYDROALCOHOLIC LEAVES EXTRACT OF LEUCAS ASPERA

The main aim of the study was to determine the phytoconstituents present in Leaves of Leucas aspera. The present study was therefore carried out to evaluate the anti-ulcer activity of Hydroalcoholic (70% methanol) leaf extract of Leucas aspera in rats. The effect of Leucas aspera extract on gastric ulcer in rats in indomethacin induced gastric ulcers model and ethanol-induced models was studied using single dosing (100, 200 mg/kg) and repeated dosing (200 mg/kg for 5 days) approaches. Ranitidine (50 mg/kg) and cimetidine (100 mg/kg) were used as the standard drugs. Depending on the model, outcome measures were volume ulcer index. Data were analyzed using one-way analysis of variance and P<0.05 was considered as statistically significant. Leucas aspera significantly (P<0.001) reduced gastric ulcer, respectively, in indomethacin induced gastric ulcers model and ethanol-induced models at the 200 mg/kg dose, which is comparable to the standard drugs. 5 days pre-treatment with Leucas aspera 200 mg/kg exhibited significant (P<0.001) ulcer inhibition. Leucas aspera possesses both dose-dependent and time-dependent anti-ulcer effect in the two models. The oral median lethal dose (LD50) is estimated to be higher than 2000 mg/kg for the crude hydroalcoholic extract, and secondary metabolites such as flavonoids, tannins, and saponins were present. The findings of this study confirmed that Leucas aspera has anti-ulcer pharmacologic activity due to one or more of the secondary metabolites present in it. Therefore, this study validates its anti-ulcer use in Ethiopian folk medicine. Further investigations on isolation of specific phytochemicals and elucidating mechanisms of action are needed.


Stomach Ulcer
Stomach ulcers, which are also known as gastric ulcers, are painful sores in the stomach lining. Stomach ulcers are a type of peptic ulcer disease. Peptic ulcers are any ulcers that affect both the stomach and small intestines. Stomach ulcers occur when the thick layer of mucus that protects your stomach from digestive juices is reduced. This allows the digestive acids to eat away at the tissues that line the stomach, causing an ulcer.
Stomach ulcers may be easily cured, but they can become severe without proper treatment.

Causes stomach ulcers
Stomach ulcers are almost always caused by one of the following:  an infection with the bacterium Helicobacter pylori (H. pylori)  long-term use of nonsteroidal antiinflammatory drugs (NSAIDs), such as aspirin, ibuprofen, or naproxen Rarely, a condition known as Zollinger-Ellison syndrome can cause stomach and intestinal ulcers by increasing the body's production of acid. This syndrome is suspected to cause less than 1 percent of all peptic ulcers.

Symptoms of stomach ulcers
A number of symptoms are associated with stomach ulcers. The severity of the symptoms depends on the severity of the ulcer.
The most common symptom is a burning sensation or pain in the middle of your abdomen between your chest and belly button. Typically, the pain will be more intense when your stomach is empty, and it can last for a few minutes to several hours.

Other common signs and symptoms of ulcers include:
 dull pain in the stomach  weight loss  not wanting to eat because of pain  nausea or vomiting  bloating  feeling easily full  burping or acid reflux  heartburn, which is a burning sensation in the chest)  pain that may improve when you eat, drink, or take antacids  anemia, whose symptoms can include tiredness, shortness of breath, or paler skin  dark, tarry stools  vomit that's bloody or looks like coffee grounds Talk to your doctor if you have any symptoms of a stomach ulcer. Even though discomfort may be mild, ulcers can worsen if they aren't treated. Bleeding ulcers can become lifethreatening.

Collection of plant material
The plant material was collected from local area of Bhopal. Drying of fresh Leaves was carried out in sun but under the shade. Dried Leaves were preserved in plastic bags and closed tightly and powdered as per the requirements.

Extraction by maceration process
65.5 gram leaves of Leucas Aspera were exhaustively extracted with hydroalcoholic extract solvent (Methanol 70%) and using drug solvent ratios (1:2) using maceration process (10hrs). The extracts were evaporated above their boiling points. Finally the percentage yields were calculated of the dried extracts (Mukherjee, 2007; Kokate, 1994).

Determination of percentage yield
The percentage yields of each extract were calculated by using following formula:

Qualitative evaluation
The chemical tests were performed for testing different chemical groups present in extracts (Khandelwal, 2005;Kokate, 1994). The Leucas aspera leaves extract acquire was subjected to the precursory phytochemical analysis following standard methods by Khandelwal and Kokate. The extract was screened to identify the presence of various active principles of Alkaloids, glycosides, Diterpenes, saponins, flavonoids and phenol.

Total Phenolic content estimation
Procedure: The total phenolic content of the extract was determined by the modified Folin-Ciocalteu method. 10 mg Gallic acid was dissolved in 10 ml methanol, various aliquots of 5-25µg/ml was prepared in methanol. 10mg of dried extract of plant material was extracted with 10 ml methanol and filter. 2 ml (1mg/ml) of this extract was for the estimation of Phenol. 2 ml of each extract or standard was mixed with 1 ml of Folin-Ciocalteu reagent (previously diluted with distilled water 1:10 v/v) and 1 ml (7.5g/l) of sodium carbonate. The mixture was vortexed for 15s and allowed to stand for 15min at 40°C for colour development. The absorbance was measured at 765 nm using a spectrophotometer.

Total flavonoids content estimation
Procedure: Determination of total flavonoids content was based on aluminium chloride method (Olufunmiso et al., 2011). 10 mg quercetin was dissolved in 10 ml methanol, and various aliquots of 5-25μg/ml were prepared in methanol. 10mg of dried extract of plant material was extracted with 10 ml methanol and filter. 3 ml (1mg/ml) of this extract was for the estimation of flavonoid. 1 ml of 2% AlCl 3 methanolic solution was added to 3 ml of extract or standard and allowed to stand for 15 min at room temperature; absorbance was measured at 420 nm.

In Vivo antiulcer activity
Preliminary experiments were carried out on rats (n=6). A Hydroalcoholic leaves extract of Leucas Aspera were administered orally in different doses to find out the range of doses which cause zero and 100 % mortality of animals. Acute oral toxicity was conducted according to the method of Organisation for Economic Co-operation and Development Based on their intensity, the ulcers were given scores as follows: 0 = no ulcer, 1 = superficial mucosal erosion, 2 = deep ulcer or transmural necrosis, 3 = perforated or penetrated ulcer.

Physical characteristics of extract
Finally extraction of defatted flowers was done with Hydro alcoholic and % yield was found to be 3.15 % w/w and their characteristics are reported in table-3.1.

Figure 4: Anti-ulcerogenic effect of Hydroalcoholic leaves extract Leucas aspera against ulcerogenic agents in rats (Ulcer index) ethanol induced gastric ulcers model
The present study investigated the effect of Hydroalcoholic leaves extract Leucas aspera on the ulcers. Hydroalcoholic leaves extract Leucas aspera showed effect on the healing of gastric ulcers induced by indomethacin. It acts through the inhibition of cell wall biosynthesis that leads to the death of the bacteria. Hydroalcoholic leaves extract Leucas aspera was effective in reducing the ulcer area and the ulcer score. Leucas aspera has an antiulcer effect. It increased healing of indomethacin induced ulcer.

Conclusion
The analysis and characterization of bioactive compounds from plants is important to ascertain their medicinal value. The phytochemical analysis showed that the Leucas aspera plant extract contains a mixture of phytochemicals as Carbohydrates, Saponins, Glycosides, Diterpenes, Phenols and Flavonoids. The total phenolics and total flavonoids content for the hydroalcoholic extract was found to be 4.27and 2.05 mg/ml in flowers extract of Leucas aspera. The present study investigated the effect of hydroalcoholic leaves extract of Leucas aspera on the ulcers. Hydroalcoholic leaves extract of Leucas aspera showed effect on the healing of gastric ulcers induced by indomethacin. It acts through the inhibition of cell wall biosynthesis that leads to the death of the bacteria.hydroalcoholic leaves extract of Leucas aspera was effective in reducing the ulcer area and the ulcer score. Leucas aspera has an antiulcer effect. It increased healing of indomethacin induced ulcer.