Phytochemical and Pharmacological Evaluation of Annona reticulate


Veena Kadasala1, Niladry Sekhar Ghosh2, Santhosh Kumar Chinnaiyan1, Arunabha Mallik3, Manjunath S.Y.1, Chiranjib Bhattacharjee1*

1Srikrupa Institute of Pharmaceutical Sciences, Vill: Velkatta, Mdl: Kondapak,

Dist: Siddipet. Telangana – 502277, India.

2AVIPS, Shobhit University Gangoh Sahranpur - 247341, Uttar Pradesh India.

3Marri Laxman Reddy Institute of Pharmacy Dundigal, Hyderabad, Telangana.

*Corresponding Author E-mail:



Annona reticulate (AR) is indigenous to the tropical areas of India and worldwide. The use of plant as remedy for diarrhea and ulcer is well documented in Ayurvedic system of medicine. However, pharmacological evidence does not exist to substantiate its therapeutic efficacy for the same. The aim was to investigate the antiulcer and anti-diarrheal activity of methanolic (ME) and aqueous extracts (AE) of A. reticulate in animal model. The antiulcer activity of extracts was investigated using ethanol and pylorus ligation-induced ulcer. The anti-diarrheal activity of MEAR and AEAR extracts was evaluated by castor oil induced diarrhea and gastro intestinal motility using parameters such as onset of diarrhea, number of wet stools, total number of stool and weight of total number of stools. The antiulcer activity of the extracts was confirmed by a reduction in ulcer index along with the decrease in gastric volume, total acidity, and an increase in pH of gastric content in both the models. A. reticulate extracts were more efficacious in reducing number of total stools in both the models of diarrhea and showed a dose-dependent antidiarrheal effect. The obtained results have established a pharmacological evidence for the folkloric use of the A. reticulate as antiulcer and antidiarrhoeal agent.


KEYWORDS: Antidiarrheal, antiulcer, pylorus ligation, Annona reticulate.




Medicinal plants continue to contribute significantly to modern prescription drugs by providing lead compounds upon which the synthesis of new drugs can be made. The 60% of the anticancer drugs and 75% of the anti-infectious disease drugs approved from 1981-2002, could be traced to natural origins. In addition, 61% of all new chemical entities introduced worldwide as drugs during the same period could be traced to or were inspired by natural products1. Pharmacologists, microbiologists, biochemist, botanists, and natural-products chemists all over the world are currently investigating medicinal plants for phytochemical and lead compounds that could be developed for treatment of various diseases2.


Unlike the ubiquitous macromolecules of primary metabolism (e.g. monosaccharides, polysaccharides, amino acids, proteins, nucleic acids, lipids) which are present in all plants, secondary metabolites with medicinal properties are found only in a few species of plants3. Some of these secondary metabolites serve as defensive compounds against herbivores and pathogens. Others function in mechanical support, in attracting pollinators and fruit dispersers, in absorbing harmful ultraviolet radiation, or reducing the growth of nearby competing plants. Secondary plant metabolites with reported medicinal properties include but not limited to polysaccharides, waxes and fatty acids, alkaloids, terpenoids, phenolics (simple phenolics and flavonoids) and glycosides and their derivatives4. Annona reticulate bark contains 0.12% anonaine, alkaloids such as Anonaine, liriodenine and reticuline (muricinine) also N-fatty acyl tryptamines5, Reticulacinone, cetogenins6, Cycloreticuline C7, Glabrin A, Cyclopeptides8. A root decoction is taken as a febrifuge, while fragments of the root bark are packed around the gums to relieve toothache9. The bark is very astringent and the decoction is taken as a tonic and also as a remedy for diarrhea and dysentery. The leaf decoction is given as a vermifuge10. The unripe dried fruit dried is employed against diarrhea and dysentery11.



Plant material:

The unripe fruits of Annona reticulata were collected from Vellikatta village, Siddipet (Telangana state) in the month of December-January. the identification and authenticated of the plant by Prof. Dr. B. Satyanarayana, Head of department in botany, Govt. Degree college (w), Siddipet, T.G.


Preliminary phytochemical screening:

The preliminary phytochemical screening was carried out on petroleum ether, chloroform, methanolic extracts, aqueous extracts of Annona reticulata for qualitative identification of type of phytoconstituents present.


Experimental animals:

Albino rats (120-200gms) and albino mice (20-30gm) of either sex were maintained under standard environmental conditions (temperature 25 ± 2°C, relative humidity 50 ± 5%) with a 12 h light/ dark cycle. Animals were fed with normal laboratory chow pellet diet and drinking water was given ad libitum. animals were allowed to acclimatize for 7 days before commencement of the experiment. All the experiments conducted on the animals were by the standards set for the use of the laboratory animal use, and the experimental protocols were duly approved by the IAEC of Srikrupa Institute of Pharmaceutical Sciences, Siddipet (IAE/ SKIPS/ 2011/ MAY 15/ 05/ RATS-72, MICE 60). 


Acute toxicity study:

The acute toxicity for methanolic and aqueous extracts of unripe fruits of Annona reticulata was determined in albino mice, maintained under standard conditions. The animals were fasted overnight prior to the experiment. Fixed dose (OCED guideline no. 420) method of CPCSEA was adopted for toxicity studies.


Antiulcer activity:

Alcohol Induced Ulcer:

Albino rats of either sex weighing between (120-200gms) were divided into six groups of six animals in each group: The animals are fasted for 24 hours with free access to water. Animals were given different extracts of Annona reticulata mentioned above of 100 and 200 mg/kg or Omeprazole 30mg/kg p.o. 1 hr later 1ml/200g of 99.80% alcohol was administered p.o. to each animal. Animals were sacrificed 1 hr. after alcohol administration, stomachs were isolated and cut open along the greater curvature and pinned on a soft board. The ulcer index was measured (0 – Normal coloured stomach, 0.5 –Red coloration, 1–Spot ulcers, 1.5–Haemorrhaegic streaks, 2– Ulcer > 3mm but < 5mm,  3–Ulcers > 5mm).


Pylorus ligated ulcer:

Albino rats of either sex body weight between (120–200gms) were divided into six groups of six animals in each group. Various extract of Annona reticulata, standard drug and control vehicle was administered 30 min prior to pyloric ligation. Under light ether anesthesia, the abdomen was opened and the pylorus was ligated. The abdomen was then sutured for 4 hr, after ligation the animals were sacrificed with excess of anesthetic ether, and the stomach was dissected out gastric juice was collected were drained into tubes and were centrifuged at 1000rpm for 10 minutes and the volume was noted. The pH of the gastric juice was recorded by pH meter. Then the contents were subjected to analysis for free and total acidity. The stomachs were then washed with running water and observed ulcer index as per mentioned in alcohol induced ulcer.


Determination of Free Acidity and Total Acidity:

One ml of gastric juice was pipetted into 100ml conical flask, added 2 to 3 drops of topfer’s reagent and titrated with 0.01 N sodium hydroxide until all traces of red colour disappears and the colour of the solution turns to yellowish orange. The volume of the alkali added was noted. This volume corresponds to free acidity. Then 2 to 3 drops of phenolphthalein solution was added and titration was continued until a definite red tinge reappears. Again, the total volume of alkali added was noted. The volume corresponds to total acidity by using the equation (1).


Acidity: (Volume of NaOH x N x100)/0.1…..(1)


Anti-diarrhoeal activity:

Castor oil-induced diarrhoea:

The method described by Galvez was used in this study. In the present study albino, mice of either sex weighing 20-25gm were used.


Gastrointestinal motility test:

The method described by O.A. Olajide was used in this study. In the present study albino mice of either sex weighing 20-25gm were used. They are divided in to 6 groups (n=6). They were fasted for 24 hrs with water ad libitum.. The percentages of distance travelled by the charcoal meal in ratio to the intestinal length and percentage of inhibition was calculated by using following equation (2 and 3).


Charcoal travel (%): (DTCM/TLSI) × 100                   (2)


Charcoal travel Inhibition (%):

(TLSI–DTCM)/TLSI×100.                                           (3)


Where, DTCM: Distance travelled by the charcoal meal, TLSI: Total length of small intestine. 

Statistical Analysis:

The results were expressed as Mean±SEM in each group. The significance of difference among the groups was assessed using one-way ANOVA followed by student t– test multiple comparison tests (Graph Pad Prism v5.0).




The extract yield, colour and consistency of various extracts of unripe fruits of Annona reticulate shown in the Table 1.


Table 1: Physical properties and extract yield of unripe fruits of Annona reticulate




Colour and Consistency

Extract yield %)


Pet. Ether

Pale Yellow Sticky




Pale Brown Solid




Very Dark Brown Solid




Dark Brown Solid




Preliminary phytochemical studies revealed that the of unripe fruits of different extracts contain flavonoids, tannins, saponins, alkaloids , glycosides and proteins in methanolic and aqueous extracts, whereas steroids are found to be present in Pet. Ether and chloroform extracts. The results are compiled in Table 2.


Table 2: Preliminary phytochemical studies of various extract of unripe fruits of Annona reticulate



Chemical Constituents








_ _

_ _

_ _




_ _

_ _

+ +

+ +



_ _

_ _

+ +

+ +



_ _

_ _

+ +




+ +

+ +


_ _



_ _

_ _

+ +

+ +



_ _

_ _

+ +

+ +

-  Absent , + Present



The acute toxicity study results have illustrated that the MEAR and AEAR administration to rats was found to be safe up to 2000 mg/kg as per the OECD guidelines 420. As no significant clinical sign like changes in the skin, eyes, mucous membranes, fur were observed in experimental animals. The reason might be due to the nontoxic nature of A. reticulate leaf extract as rat have fully tolerated and has neither the water nor the food intake was reduced during the study period. Further, it was observed that no mortality has occurred in any of the groups with all the dosages.



Ethanol induced ulcer:

MEAR and AEAR 200 mg/kg showed significant dose-dependent protection in ethanol-induced ulcer. The percentage protection values were 82.52, 82.21 (p < 0.01). Ethanol-induced gastric ulcers in animals showed extensive lesions that were restricted to glandular portions of the stomach as compared to control rats (P < 0.05), whereas oral administration of MEAR and AEAR lowered the U.I in Group IV and Group VI Table 3.


Table 3: Effect of methanolic and aqueous extract of unripe fruits of Annona reticulata on ethanol induced ulcer in rat

S. No


Ulcer index

% of ulcer protection


Control D.W








MEAR1(100 mg/kg)




MEAR2 (200 mg/kg)




AEAR1 (100 mg/kg)




AEAR 2 (200 mg/kg)



Values are the mean ± S.E.M., (n=6), significant at *P < 0.05 and **P < 0.01, When compared to control group. Standard Drug; Omeprazole (30mg/kg). D.W: Distilled water.


Pylorus ligated ulcer:

A reticulate extracts also showed antiulcer activity by reducing the volume of gastric juice and acid output significantly (P < 0.05). When compared with the control group of rats, pre-treated with ME at the doses of 100mg/kg, p.o. and 200mg/kg, p.o. dose-dependently and significantly (P < 0.05) inhibited the volume of gastric juice, acid output, and U.I [Table 4]. There was also a significant (P < 0.01) increase in pH of gastric content in the presence of MEAR when compared with the control. The standard drug Omeprazole (30mg/kg, p.o.) and the 100 and 200mg/kg doses of the extract produced a significant reduction in U.I, volume gastric juice, and acid output Table 4.


Table 4: Effect of methanolic and aqueous extract of unripe fruits of  Annona reticulata on Pylorus-ligated rats


Gastric content



free Acidity (meq/L)

Total acidity (meq/L)

Ulcer Score

Mean Ulcer index

% of Ulcer protection

Control D.W










2.86 ±0.18**






MEAR1(100 mg/kg)


2.21 ±0.27*






MEAR2 (200 mg/kg)


2.48 ±0.27*






AEAR1 (100 mg/kg)


2.1 ±0.18*






AEAR 2 (200 mg/kg)


2.35 ±0.30**






Values are the mean ± S.E.M., (n=6), significant at *P < 0.05 and **P < 0.01, When compared to control group. Standard Drug; Omeprazole (30mg/kg). D.W: Distilled water



Table 5: Effect of methanolic and aqueous extracts of unripe fruits of Annona reticulata  on castor oil induced diarrhoea


onset time of diarrhoea (min)

Total no. of faeces

No.  of wet faeces

Total weight of wet faeces (mg)

Control D.W










MEAR1(100 mg/kg)





MEAR2 (200 mg/kg)





AEAR1 (100 mg/kg)





AEAR 2 (200 mg/kg)





Values are the mean ± S.E.M., (n=6), significant at *P < 0.05 and **P < 0.01When compared to control group. Standard drug; Loperamide (3 mg/kg)



Castor oil induced diarrhea:

After administration of castor oil, all the rats in the control groups produced copious diarrhea. The MEAR and AEAR on oral administration at doses of 100 and 200mg/kg to rat reduced in a dose-dependent manner the frequency of feces as well as the wet weight of fecal droppings when compared with untreated control significantly (P < 0.01). The observed experimental results are presented in Table 5.


Gastrointestinal motility:

Pre-treatment with MEAR and AEAR have reduced the GIT motility in a dose dependent manner by the reduction in the movement charcoal meal. Between the two extracts methanolic extract (**P < 0.01) was found to be most potent Table 6.


Table 6: Effect of methanolic and aqueous extracts of unripe fruits of Annona reticulata  on Gastrointestinal motility.




Distance traveled by charcoal (%)








MEAR1 (100 mg/kg)



MEAR2 (200 mg/kg)



AEAR1 (100 mg/kg)



AEAR 2 (200 mg/kg)


Values are the mean ± S.E.M., (n=6), significant at *P < 0.05 and **P < 0.01When compared to control group. Standard Drug: Atropine sulphate (i.p)



Peptic ulcer is one disease, which required treatment for chronic period. The usage of allopathic drugs for such a long time may results in adverse effect, adverse reaction, drug interactions etc13. Therefore, several traditionally used drugs are being verified for this purpose and are available in the market for the purpose. In the present study one more herbal drug A. reticulata which was used traditionally for treating ulcer and diarrhea. The reported studies claimed that the unripe fruits were used for ulcers and diarrhoea. Polyphenolic compounds were known to have anti-oxidant property and anti-oxidants are having gastroprotective role against various experimentally induced ulcer with a intention of verifying the claims of a native practitioner and correlate the results with the earlier reports 14-16.


The methanolic and aqueous extracts of unripe fruits were subjected for screening anti-ulcer activity by using the following models. (i) Ethanol induced gastric ulceration, (ii) Pylorus ligation induced gastric ulceration. The parameters of the study were the reduction in the ulcer index in all the models and reduction in volume of secretion and acidity and increase in gastric pH in pylorus ligation model. In ethanol induced gastric ulceration model, ethanol (1 ml/200 g), has induced severe ulcers as indicated by increase in red colouration, number of red spots, hemaorrhagic streaks and larger ulcers. There are reports that alcohol induces ulcer are due to increase secretion of proteins in to the gastric juice, reduced GSH and GPH levels in gastric mucosa, in addition ethanol causes ulcers by producing toxic oxygen derived free radicals17. In the present study treatment with MEAR and AEAR extracts of unripe fruits have shown significant and dose dependant gastric protective activity against ethanol induced ulcer as well as pyloric ligation method. Both extracts have been showed almost similar activities. The gastro-protective activity against ethanol induced ulcers may be attributed to the anti-oxidant principle, probably the flavonoids and tannins18. Since the extracts have shown significant anti-diarrhoeal property against castor oil induced diarrhoea, the gastro-protective activity of the extract may not be due to interfering with the arachidonic acid metabolism, it seems here also inhibition of back diffusion of H+ ions due to aspirin may be the possible mechanism of gastro-protective action of these extracts19.  In the pyloric ligation model the elevation in the gastric secretion and the ulcer may be due to the in balance between aggressive factors and mucosal integrity maintain by endogenous defense mechanisms. Several studies also indicated that prostaglandin may acts as gastro-protective as well as decrease in acid and pepsin secretion20. This increased secretion of acid and pepsin may lead to auto-digestion of gastric mucosa and break down of mucosal barrier. In addition pylorus ligation may decrease GSH contain in gastric mucosa and increase mucosal lipid peroxidation21. In the present study, acid secretion was decrease and gastric pH was raised but there are reports that pepsin acts only at lower pH. Since there was an elevated pH, the pepsin becomes inactive and thereby there is a reduction in digestion of mucosal barrier. Since there is a report that the lipid peroxidation is increase due to pylorus ligation and lipid peroxidation is due to free radicals. Overall, our results were indicating that two extracts of the plants of the present study are possessing gastro-protective activity. It appears that gastro-protective activity may be due to anti-oxidant property of plant and this anti-oxidant activity may be attributed to polyphenolic compound (flavonoids and tannins) of the plants.


The plant is highly useful in treating diarrhoea, MEAR and AEAR of unripe fruits of Annona reticulata were subjected to screening for anti-diarrhoeal activity by using following experimentally induced diarrhoeal models. (i) Castor oil induced diarrhea, (ii) Gastrointestinal motility in mice. In castor oil induced diarrhoea model, administration of castor oil (0.5ml) increase the total number of faeces, number of wet faeces and total weight of wet faeces and these was an decrease in time elapsed between the administration of the cathartic agent and the excretion of fist diarrhoeal faeces. This was reversed by the pre-treatment with MEAR and AEAR of unripe fruits, significantly in a dose dependant manner. In castor oil induced diarrhoeal models there are reports that castor oil increases peristatilic activity and produce permeability changes in intestinal membrane to electrolytes and water22-26. It seems methanolic and aqueous extracts of unripe fruits of the plant have reduced the diarrhoea induced by castor oil against reversal of enhanced peristatilic activity and change permeability to electrolyte and water. However the exact mechanism could not established. In our results MEAR was found to be most potent in reversing the castor oil diarrhoea. There were reports that tannins possessed anti-diarrhoeal property27-29. Probably tannin content of the plant was responsible for anti-diarrhoeal activity against castor oil induced diarrhea30-31. The charcoal meal method treatment with MEAR and AEAR of unripe fruits have significantly decreased the GIT motility in dose dependant manner and which was comparable with standard atropine. Out of all the MEAR was most potent and it seems tannins have a role in anti-diarrhoeal property.



The preliminary phytochemical studies show that the presence of steroids, alkaloids, saponin, flavonoids and tannins in unripe fruits of methanolic and aqueous extracts of the plant, studied. The methanolic and aqueous extracts of unripe fruits showed the presence of flavonoids and tannins and selected for the study. In Alcohol and Pylorus ligation induced ulcer models, both extracts showed significant gastroprotective activity in a dose dependant manner. They also reduces gastric volume, total acidity, free acidity of gastric juice and increase the pH of gastric juice, so we can conclude that both extracts of the plant showed anti-secretory and gastroprotective property. Both the methanolic and aqueous extracts of the plant possess anti-diarrhoeal property as indicated by the reduction in the gastric motility, total weight of wet faeces, in all the experimental models. Between two extracts, methanolic extract produced most potent anti-diarrhoeal activity.



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Received on 29.06.2020           Modified on 07.09.2020

Accepted on 19.10.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(9):4881-4886.

DOI: 10.52711/0974-360X.2021.00848