Phytochemical and Antiulcer potential of Ethanolic leaf extract of Heliotropium indicum Linn.

 

Basak M*, Saha S

Faculty of Pharmaceutical Sciences, Assam Down Town University, Panikhaiti, Guwahati, Assam - 781026

 

ABSTRACT:

In the present study Phytochemical, Pharmacological studies were performed. Heliotropium indicum Linn. belongs to family Boraginaceae and commonly known as Leihenbi, Hatisur, devil weed. The leaves have been used as asthma, urticaria, amenorrhea, high blood pressure, fevers, ulcers, and cough etc. The present study includes ethanolic leaves extract of Heliotropium indicum Linn. were investigated for phytochemical screening. The leaves of Heliotropium indicum Linn. were collected from different part of Meghalaya and Assam and cut into small pieces and shade dried. The dried powdered leaves (100gm) were extracted in a Soxhlet apparatus by using different solvent by increasing polarity order. Phytochemical screening has been done by standard procedure and showed the presence of alkaloids, glycoside, carbohydrate, flavonoids, and saponins. Gastric ulcer is one of the most prevalent gastrointestinal disorders, which affects approximately 5-10% of people during their life. In recent years, abundant work has been carried out on herbal medicine to clarify their potential efficacy in gastric ulcer prevention or management. Here, the present study was carried out to investigate the antiulcer activity of ethanolic leaf extract of Heliotropium indicum Linn. in aspirin plus pylorus ligated ulceration in the albino rats. Two dose levels of the extracts i.e. 250 and 500mg/kg were selected for the further study. In Aspirin plus pylorus ligation induced ulcer model, various parameters were studied viz. gastric volume, pH, total acidity, free acidity, and ulcer index. Ranitidine at 50mg/kg was used as the standard drug. Pre-treatment of ethanol extract of Heliotropium indicum Linn. leaves showed a significant decrease in the gastric volume, total acidity, and free acidity. However, pH of the gastric juice was significantly increased only at a higher dose, 500 mg/kg. It showed also a significant decrease in a number of ulcers and ulcer score index in aspirin plus pylorus ligation induced ulceration models. The ethanol extract of Heliotropium indicum Linn. leaves possess significant antiulcer properties in a dose dependent manner. In conclusion, the antiulcer properties of the extract may be attributed to the presence of phytochemicals like flavonoids, alkaloids and tannins present in the plant extract with various biological activities. Present work justifies the traditional use of Heliotropium indicum Linn. in treatment of the diverse condition and also indicated its future importance as therapeutic substances.

 

 

 

 

INTRODUCTION:

Peptic ulcer ailment is portrayed by aroused sores or unearthings of the mucosa and fundamental tissue of the upper gastrointestinal tract. The ulcers are the aftereffect of harm to the bodily fluid film which typically secures the throat, stomach, and duodenum from gastric corrosive and pepsin.¹ The pathophysiology of this gastro-intestinal disorder is due to an imbalance between mucosal defensive factors such as bicarbonate, prostaglandin, nitric oxide, peptides, growth factors and injurious factors like acid, pepsin.² Gastric ulcers are often a chronic disease and may persist for 10-12 years characterized by a repeated episode of healing and exacerbations.³

 

A peptic ulcer is the most common, chronic gastrointestinal disorder and has become a common global health problem affecting a large number of people and a major cause of morbidity and mortality.⁴ Geographically, the disease is prevalent throughout the world, in USA annually 3.7 million people are affected by this disease.

 

Drugs employed for the treatment of peptic ulcer have three therapeutic approaches (i) to relieve pain (ii) to hasten healing and (iii) to prevent ulcer reappearance. These three therapeutic approaches are helpful to reduce aggressive forces by lowering H+ output, to increase protective forces by means of mucoprotectants and to eradicate H. pylori.^5,6

 

Heliotropium indium Linn. are rich in pyrrolizidine alkaloids. The aerial parts contain echinatine, heleurine, lasiocarpine-N-oxide, supinine, heliotrine, indicine, indicine-N-oxide, and lasiocarpine. Presence of cynoglossine, europine-N-oxide, heleurine-N-Oxide, heliotridine-N-Oxide, heleotrine-N-Oxide and heliotrine has been identified from the seeds. Other alkaloids such as putrescine, spermidine, homo spermidine and spermine have been identified in the leaves. Apart from alkaloids, several triterpenes and steroids like lupeol, chalinasterol, stigmasterol and campesterol are found in entire plant. Other compounds reported are rapanone and hexacosan-1-ol and presence of estradiol in the roots. Helindicine, a new pyrrolizidine alkaloid together with the known lycopsamine were isolated from the roots of Heliotropium indicum, Linn. The structures established by a combination of 1D and 2D NMR methods (COSY, HMQC, HMBC, and NOESY) and HREIMS. This first reported was lactone pyrrolizidine alkaloid in the genus Heliotropium. Helindicine and lycopsamine were assayed for antioxidant activity and has moderate activity. In another experiment, the volatile oil isolated by hydrodistillation was analysed by a combination of GC–FID and GC–MS. The major constituents of the volatile oil were phytol (49.1%), 1-dodecanol (6.4%). and β-linalool (3.0%).^7,8.

 

In the Philippines, decoction of leaves are used for washing cuts and sores; Also used for the treatment of cholera and antiscabious. Poultice of pounded leaves or bruised leaves are applied to wounds and boils and in inflamed joints. Juice of leaves is used for facial acne, gum-boils, sores and wounds.

 

Sap of leaves are mixed with salt and used for clearing vision. Plant used for ear and skin infections. Leaves used for treatment of herpes and rheumatism. Decoction of leaves and flowers are used as gargle for sore throats and tonsillitis. Flowers are used in small doses are emmenagogue; in large doses, abortive practices. In India, juice of leaves used for treating sores and insect bites. Decoction of leaves also used for urticaria. In Nigeria, plant is used for fevers and ulcers. In Senegal, leaf powder used for dermatitis, eczema, impetigo in children. The plant also used for wound healing in Thailand. In Taiwan, a folk remedy for sore throat and lung diseases. In Nigeria, an infusion of the plant is used as an eye-lotion and to clean ulcers. In Madagascar a plant infusion is used as a strong diuretic. ^9,10

 

MATERIAL AND METHODS:

Collection and Identification of the Plant materials:

The plant Heliotropium indicum Linn. was collected in the month of March-may from lower Assam of Assam. The plant was identified from the standard literature  then confirmed and authenticated by Dr. A.A. Mao (Scientist-E and H.O.O), Botanical Survey of India (BSI), Eastern Regional Centre, Shillong- 793003 (Ref No: BSI/ERC/2015/Plant identification/151). The collected plant material were washed thoroughly with the help of water to remove the earthy matter or adherent impurity and then shade dried .The dried material were powdered by means of mechanical grinder. The subsequent powdered material was put away in hermetically sealed glass holder for further studies.

 

Experimental animals:

Healthy young Albino rats weighing between 120g to 200g were procured from the Assam down town University animal house. The animals were individually housed in polypropylene cage and the room condition was maintained at temperature of 25±5ºC and humidity 45±5 per cent with 12 hr day and night cycle. The animals were fed with Pellet chew feed standard diet and water ad libitum. Every trial methodology were directed with the endorsement of the Institutional Animal Ethics committee CPCSEA (Reg. No- AdtU/IAEC/2015/006) for the care and use of animals and their guidelines were strictly followed throughout the study.

 

Phytochemical Screening:

The extracts obtained within the sequent solvent extraction method were then subjected to numerous qualitative tests according method^11,12 to work out the presence of assorted phytoconstituents like alkaloids, glycosides, saponin, flavonoids, carbohydrates, amino acids, sterols, gums and mucilage etc.

 

The concentrated extracts were subjected to chemical test as per the methods mentioned below for the identification of the various constituents.

 

1. Detection of alkaloid:

About 50mg of solvent free extract was stirred with few ml of dilute hydrochloric acid and filtered through filter paper. The filtrate is tested carefully with various alkaloidal reagents as follows:

·       Mayer’s test:

To a few ml of filtrate, a drop or two of Mayer’s reagent were added by the side of the test tube. A white or creamy precipitate indicates test as positive.

·       Wagner’s test:

To a few ml of filtrate, few drops of Wagner’s reagent were added by the side of the test tube. A reddish-brown precipitate indicates test as positive.

·       Hager’s Test:

To a few ml of filtrate 1 or 2ml of Hager’s reagent were added by the side of the test tube. A prominent yellow colour precipitate indicates  as a  positive test.

·       Dragendorff’s Test:

To a few ml of filtrate, 1 or 2ml of Dragendorff’s reagent were added by the side of the test tube. A prominent yellow precipitate indicates test as positive.

 

2. Detection of carbohydrates:

The extract (100mg) is dissolved in 5ml of water and filtered. The filtrate is subjected to the following test.

·       Molish’s Test:

To 2ml of filtrate, 2 drops of alcoholic solution of alpha-naphthol were added, the mixture is shaken well and 1ml of conc. Sulphiuric acid was added slowly along the side of the test tube and allowed to stand. A violet ring indicates the presence of carbohydrate.

·       Fehling’s Test:

1ml of filtrate was boiled on water bath with 1ml each of Fehling solution A and Fehling solution B; a red precipitate indicates the presence of sugar.

·       Benedict’s Test:

To 0.5ml of filtrate, 1ml of Benedict’s reagent was added. The mixture was heated on a boiling water bath for 2 min., a characteristic colored precipitate indicates the presence of sugar.

·       Barfoed’s Test:

To 1ml of filtrate add ml of Barfoed’s reagent and heated on a water bath for 2 min. and if red precipitate occurs it indicates presence of sugar.

 

3. Detection of saponins:

The extract (50mg) is diluted with distilled water and made upto 20ml. The suspension is shaken for 15 min. A layer of 2cm of foam indicates the presence of saponins.

 

 

4. Detection of phenolic compounds:

·       Ferric Chloride Test:

The extract (50mg) was dissolved in 5ml of distilled water. To this few drops of natural 5% ferric chloride solution was added. A dark green color indicates the presence of Phenolic compounds.

 

 

·       Gelatin Test:

About 50mg of extract was dissolved in 5ml of distilled water and 2ml of 10% sodium chloride solution was added. White precipitates indicate the presence of Phenolic compounds.

 

5. Detection of glycoside and flavonoids:

50gm of extract was hydrolyzed with concentrated hydrochloric acid for 2hr on a water bath, filtered and the hydrolyses is subjected to the following test:

·       Borntrager’s Test: 

2ml of filtered hydrolyses was taken and add 3ml of chloroform and shaken, chloroform layer was separated and 10% amonia solution was added to it if pink color occurs it indicates the presence of glycosides.

·       Lead Acetate Test:

The extract (50mg) was dissolved in distilled water and to this; 3ml of 10% lead acetate solution was added. If bulky white precipitate occurs, the presence of Phenolic compounds.

·       Magnesium and Hydrochloric Acid Reduction:

The extract (50mg) was dissolved in 5ml of alcohol and few fragment of magnesium ribbon and conc. Hydrochloric acid acid (drop wise) was added. If any pink to crimson color develops, presence of flavanol glycosides is inferred.

·       Alkaline Reagent Test:

An aqueous solution of the extract was treated with 10% Amonium hydroxide solution. Yellow fluorescence indicates the presence of flavonoids.

·       Aqueous Sodium Hydroxide Test: 

An aqueous solution of the extract was treated with sodium hydroxide solution it gives blue to violet (anthrocyanins), yellow (flavones), and yellow to orange (flavonones).

·       Concentrate Sulphuric Acid Test: 

An aqueous solution of the extract was treated with concentrate sulphuric acid it gives yellowish orange (anthrocyanins), yellow to orange (flavones), orange to crimson (flavonones).

 

6. Detection of proteins and amino acids:

The extract (100mg) is dissolved in 10ml of distilled water and filter through whatman filter paper no-1 and the filtrate was subjected to tests for proteins and amino acids.

·       Millon’s Test:

To 2ml of filtrate, few drops of Millon’s reagent were added. A white precipitate indicates the presence of proteins.

·       Biuret Test:

An aliquot of filtrate was treated with one drop of 2% copper sulphate solution. To this 1ml of ethanol (95%) is added, followed by excess of potassium hydroxide palate. Pink color in the ethanolic layer indicates the presence of proteins.

·       Ninhydrin Test:

To 2ml of aqueous filtrate add two drops of ninhydrin solution (10mg of ninhydrin in 200ml of acetone). A characteristic purple color indicates the presence of protein.

·       Legal’s Test:

50mg of extract was dissolved in pyridine; sodium nitroprusside solution was added and made it alkaline using 10% sodium hydroxide. A characteristic pink colour indicates the presence of glycoside.

·       Keller-Killiani Test:

To an extract of drug in glacial acetic acid, few drops of ferric chloride and conc. Sulfuric acid were added. A reddish-brown color was formed at the junction of two layers and the upper layer turns bluish green.

 

7. Detection of phytosterols:

·       Liebermann- Burchard’s Test:

To 50mg of extract 2 ml acetic anhydride was added. To this solution 1-2 drops of conc. sulfuric acid was added, along the sides of the test tube. An array of color changes shows the presence of phytosterols.

 

8. Detection of fixed oils and fats:

·       Spot Test:

Press a small quantity of extract separately between two filter papers. Oil stains on the paper indicate the presence of fixed oil.

·       Saponification Test:

Add some drops of 0.5 N alcoholic potasium hydroxide to a little amount of extract beside a drop of phenolphthalein. Heat the mixture on water bath for 1-2 hr. Formation of soap or partial neutralization of alkali indicates the presence of fixed oils and fats.

 

9. Detection of gums and mucilage

About 100mg of extract was dissolved in 10ml of distilled water and to this 25ml of absolute alcohol was added with constant stirring. White or cloudy precipitate indicates the presence of gums and mucilage.

 

10. Detection of coumarin:

About 50mg of extract was dissolved in 10ml absolute alcohol and to this few drops of ferric chloride is added. Greenish fluorescence indicates the presence of coumarin.

 

Pharmacological studies:

Acute toxicity study:

Acute toxicity for extract will be determined in Albino mice as per OECD Guideline no. 423, Annexure 2d method. The tested extracts will be suspended in 0.5% w/v sodium carboxy methyl cellulose (CMC) and were administrated orally (1ml/100g) in 3 animals. The presence or absence of any signs of toxicity or mortality was monitored at 2000mg/kg in the all cases. Common side effects such as, mild diarrhea, lose of weight and depression of treated groups of animals were recorded within the 7 days of observation.

 

Antiulcer activity:

Aspirin plus pylorus ligation-induced ulcer:

Ethanolic leaf extract, aspirin and ranitidine were prepared in 0.5% carboxymethyl cellulose (CMC) as suspension and administered orally once daily at a volume of 10ml/kg for 7 days using an oral gavage needle. The rats were divided into seven groups (n=6). Group I was administered with 0.5% CMC, which served as normal control. Group II received only aspirin (200mg/kg) and served as ulcer control group, group III, IV, V and VI were treated with leaf extracts (HC and HI ethanolic extracts 250 and 500mg/kg, respectively). Group VII received ranitidine (50mg/kg) and served as standard.

 

From day 5 to day 7, rats in group III-VII received aspirin at a dose of 200mg/kg, 2hr after the administration of the respective drug treatment. Rats in all the groups were anaesthetized with ether on day 8 after 18 hr of fasting. The guts was cut opened by a little midline entry point beneath the xiphoid procedure and pylorus bit of the stomach was lifted out and ligated maintaining a strategic distance from footing to the pylorus or harm to its blood supply. At the end of 4 hr after ligation, the rats were scarified with excess of anesthetic ether and the stomach was dissected out.¹³

 

Measurement of ulcer index: 

The mucosa was flushed with saline and stomach stuck on a frog board. The lesion in glandular portion examined under a 10x magnifying glass and length was measured using a divider and scale and gastric ulcer was scored. Ulcer index of each animal was calculated by adding the following values and their mean values determined¹⁴

·       Loss of normal morphology - 1

·       Discolouration of mucosa - 1

·       Mucosal oedema - 1

·       Haemorrhages - 1

·       Petechial point (until 9) - 2

·       Petechial point (>10) - 3

·       Ulcer upto 1 mm - nX2

·       Ulcer >1 mm - nX3

·       Perforated ulcer - nX4

 

Where, ‘n’ is the number of ulcer found.

 

Percentage inhibition of ulceration was calculated as below:

                                UI ulcer control - UI treated

% Inhibition =      –––––––––––––––––––––––––– × 100

                                     UI ulcer control

 

Biochemical parameters:

The stomach was carefully excised keeping esophagus closed and opened along the greater curvature and luminal contents were removed. The gastric substance were then gathered in a test tube and centrifuged. The samples were analyzed for gastric juice volume, pH, free and total acidity.

 

Measurement of gastric juice volume and pH:

Gastric juice was collected from aspirin plus pylorus ligation induced ulcer rats. The gathered gastric juice was centrifuged at 3000rpm for 10 min. The volume of supernatant was estimated and expressed as ml/100g body weight. The pH of the supernatant was estimated utilizing advanced pH meter.¹⁵

 

Determination of free and total acidity:

An aliquot of 1.0ml of gastric juice was pipetted out into a 50.0ml conical flask and 2/3 drops of Topfer’s reagent was added to it and titrated with 0.01 N NaOH until all traces of the red colour disappeared and the colour of the solution turned yellowish orange. The volume of 0.01 N NaOH noted. The volume corresponds to free acidity. After that 2/3 drops of phenolphthalein reagent was added and then titration was continued until a permanent pink color was developed. The volume of total alkali consumed was noted which corresponds to total acidity. The free acidity and total acidity was determined using the formula and values are expressed as meq/l per 100 g.¹⁶

 

              Volume of NaOH × Normality of NaOH × 100

Acidity = ––––––––––––––––––––-–––––––––––––– (meq/l per 100 g)

                                                    0.1

 

RESULTS AND DISCUSSION:

In the present study an attempt has been made to evaluate the phytochemical and pharmacological studies of the leaves of Heliotropium indicum Linn.

 

The preliminary phytochemical screening of the crude extract of leaves of Heliotropium indicum Linn.. were carried out in order to ascertain the presence of its constituents such as glycosides, tannins, carbohydrate, protein amino acids, alkaloids, carbohydrates, flavonoids and coumarins (Table no 1).

 

In our antiulcer study, a comparison was made between the leaf extracts of above-mentioned plants with the well-established first line anti-ulcer drug ranitidine in aspirin induced and pylorus ligation induced ulcer in rats. Aspirin interfere prostaglandin synthesis via cyclooxygenase pathways and produce neutrophil and oxygen radical dependent micro-vascular injury in prolong use which ultimately leads to mucosal damage.^17-19 According to another explanation, aspirin directly increases the H+ particle transport on the mucosal epithelial cells and reductions mucin, surface-dynamic phospholipids, bicarbonate discharge.²⁰ In pylorus ligated rats accumulation of gastric acid and pepsin secretion are important factors for generation of ulcer.

 

When aspirin was administered to pylorus ligated rats, extensive damage on the glandular region of the stomach was observed. This result is quite obvious due to the cumulative effect of ulceration property of aspirin and pylorus ligation.^21,22

 

Treatment with test drug i.e. leaf extract decreased the ulcer index compared to ulcer control group, which is the direct indication of its antiulcer activity (Table No. 2 and Fig no. 1, 2, 3, 4, 5). Test drugs treatment also decreased volume of acid secretion, peptic activity, total and free acidity and increased pH of the gastric juice compared to ulcer control group proving the antiulcer activity of the extracts (Table No. 3)

 

Table-1 Preliminary phytochemical analysis of leaf of Heliotropium indicum Linn.

(+)= Present   (-) = Absent

 

Table-2 Effect of ethanolic extract of Houttuynia cordata, Thumb and Heliotropium indicum Linn on ulcer index in the stomach of aspirin plus pylorus ligated rats

Values are expressed as (Mean ± S.E.M.), n= 6, **p<0.01 and ***p< 0.001 when compared with control group. (Statistically analyzed by one-way analysis of variance (ANOVA) followed by Tukey test.)

 

 

Table-3 Effect of Houttuynia cordata, Thumb and Heliotropium indicum Linn extract on gastric parameters in aspirin plus pylorus ligation-induced ulcer rats

Values are expressed as (Mean ± S.E.M.), n= 6, **p<0.01 and ***p< 0.001 when compared with control group. (Statistically analyzed by one-way analysis of variance (ANOVA) followed by Tukey test.)

 

Fig- 1 Stomach of healthy control group (0.5% CMC)    Fig- 2 Stomach of disease control group Aspirin (200 mg/kg)

 

 

 

Preliminary phytochemical analysis of the above-mentioned leaf extract revealed the presence of alkaloids, flavonoids, saponin glycosides and phenolic compounds as active compounds. It has been well established that the above-mentioned active compounds especially flavonoids are showing cyto-protective activity by stimulating mucus, bicarbonate and prostaglandin secretion and inhibiting the neutrophil and oxygen radical dependent micro-vascular injury in GI lumen.²³ So the antiulcer activity of Heliotropium indicum Linn. may be attributed to its flavonoids content. The results of the present study suggest that the ethanolic extract of Heliotropium indicum Linn. leaves might be valuable in the treatment of gastric sores. Additionally, concentrates to distinguish the dynamic moieties and clarification of the component of activity are suggested.

 

SUMMARY AND CONCLUSION:

In the present work Phytocemical screening and Pharmacological studies has been performed. The quantitative phytochemical analysis has shown the presence of flavonoid, glycoside, alkaloid, carbohydrate, phenolic compounds and coumarin in different extracts which obtained through successive solvent extraction Prevalence of peptic ulcer disease is reported as one of the major diseases. Alcohol consumption is one of the reasons for the disease. From the present animal model study, it is proved that Heliotropium indicum Linn. ethanolic leaf extract is a potent antiulcer and gastro-protective agent. Treatment with this plant extracts in ulcer induced rats reduced the offensive factors such as ulcer index and total acidity of gastric juice. It was proved that the performance of Heliotropium indicum Linn. leaves extract showed good results to standard drug, Ranitidine.

 

The result of the study has seen to provide support for the use of Heliotropium indicum Linn. as antiulcer agent in the traditional medicine system. To promote proper conservation is sustainable use of such plant resources, awareness of local communities should be enhanced incorporating the traditional knowledge with scientific findings.

 

ACKNOWLEDGEMENT:

The authors are grateful to the authorities of Assam down town University for the facilities.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 29.01.2019            Modified on 11.12.2019

Accepted on 05.06.2020         © RJPT All right reserved