INTRODUCTION:

The illness known as Functional gastrointestinal disorder is associated with chronic gastrointestinal issues, and is becoming prevalent in the population of urban areas. In FGID there are many diseases like irritable bowel syndrome (Spastic bowel), functional dyspepsia, gastroesophageal reflux (Acid reflux) diseases and so constipation, diarrhoea, bloating, abdominal pain, indigestion, nausea etc.¹

Functional gastrointestinal disorder is caused by variable factors includes genetics, smoking, stress, and the motility of the gut. Because of the physiology entailed and the connection with the word functional, these illnesses have been renamed disorders of brain-gut interaction to more accurately represent their foundation which is scientific².

Due to the belief that medications derived from natural sources are more beneficial, harmless than those derived from synthetic sources, there has been a worldwide resurgence of interest in herbal remedies³. The latex found in many plants and latex-containing many constituents which help in the treatment of many diseases like cancer, viral disease, wound healing, convulsion etc.⁴ Tabernaemonatna is a genus of 100-120 flowering plants in the Apocynaceae family. Tabernaemonatana divaricata is used in Ayurveda, countries like China and uses traditional medicine to treat high fever, pain, inflammation, and diarrhoea. Tabernaemonatana divaricata is also known as Chandani or Crepe Jasmine and is a member of the Apocynaceae family. T. divaricata contains proteases which are responsible for the treatment of FGID⁵. At the same time, the scientifically most advantageous herbal formulation in gastrointestinal diseases is proven to be nanoemulsions⁶. Because of all these observations in the literature, the present study targeted to evaluate the potentiality of the nanoemulsion formulation of T. divaricata for the overall treatment of FGID.

MATERIAL AND METHODS:

Collection and authentication of plant:

Fresh plant of T. divaricata was purchased and collected from Pragati Garden Nursery, Chhattarpur, New Delhi. After collection, the plant was authenticated by the Botanic Garden of the Indian Republic, Gautam Buddha Nagar, Noida- 201303. After the authentication of the plant, the protease was isolated from the latex of the plant. and it was then made into crystal which was then preserved in the refrigerator for further use.

Preparation and extraction:

Protease was isolated from the latex of the plant T. divaricata. Latex was obtained from the stem and flowers of T. divaricata and placed in glass tubes with 1 ml of 10 percent sodium metabisulphite. The latex was frozen and thawed repeatedly before being centrifuged at seven thousand RPM at 5 degrees centigrade for thirty minutes to remove suspended coarse inert elements. The crude enzyme was made from the supernatant and stored at 4 degrees Celsius⁷.

Experimental Animals:

Wistar rats of weight 150-200gm. and Swiss albino mice (any sex) of weight 25-30gm. were utilized in this experiment. The protocol for an animal experiment (IAEC/NIET/2022/01/06) has been approved by the IAEC of Noida Institute of Engineering and Technology (Pharmacy Institute), Greater Noida. Animals were obtained and procured in the animal house at the Noida Institute of Engineering and Technology (Pharmacy Institute) for 90 days under standard conditions, which comprised a temperature of 24^oC, a 12:12 hr. cycle for dark and light.

Acute toxicity study:

The proteases which were isolated from the latex of T. divaricata was tested for acute oral toxicity using the OECD guideline no 423. The investigation was conducted using mature Swiss albino mice weighing 25-30gm in good health. 4 groups of three animals each were formed, and the animals fasted for one night. The doses for groups I, II, III, and IV were 5, 50, 300, and 2000mg/kg respectively. Several measures used such as body temperature, CNS activity, deification, etc., were measured after protease administration. After administration protease was checked the toxicity up to 14 days but there was no toxicity on the higher dose of 2000mg/kg. So below 2000mg/kg dose was considered an effective dose. After completing the oral toxicity study, I chose 2 doses of protease one was a lower dose of 500mg/kg, and one was a higher dose of protease of 1000mg/kg⁸.

Preparation of Nanoemulgels of protease from the latex of T. divaricata (NPLT):

The casein nanogel was prepared by following the emulsion-solvent diffusion method. As per the considered dose (500 and 1000mg/Kg) of the protease from the latex of T. divaricate was weighed accurately and so ethyl cellulose was dissolved in dichloromethane. Whereas, Carbopol 934 was used to prepare the aqueous phase by stirring and heating continuously with addition of tween 80. Further, the phase with the drug was added into the aqueous phase with homogenization for emulsification. The hydrogel was obtained by adding triethanolamine to the gel to maintain the pH.^9-11

Evaluation of Nanoemulgels:

Physical Appearance: The formulated nanogels were visually detected for their appearance, presence of aggregates, colour and homogeneity after allowing the gel to settle in the container. They were then tested to check the presence of any aggregates.^9-12

pH measurement: The pH of NPLT was checked with Digital pH meter (Syntronics, India) by dipping the glass electrode into the aqueous nanogel. It was carefully monitored to prevent any irritation to the nasal mucos.^9-12

Spreadability: To know the extent of surface area NPLT spreads on its application to the skin, between two horizontal planes the gel is placed and a 200gm (M) standard weight is applied to the top plate. After 1 min (T) the spreading diameter (L) was measured. The spreadability (S) was measured following the formula

S= M*L/T¹².

Particle size: By following proton correlation spectroscopy, the particle size of the nanoemulgel was recorded using a Zeta sizer (Malvern aster sizer 2000 MS). In distilled water, 1 ml nanoemulgel was diluted and till get a clear solution. The zeta potential and droplet size were determined to get idea about its absorption rate.^9-12

Evaluation of NPLT for Functional Gastrointestinal Disorder (FGID):

Food-related model of FGID:

Food plays an important role in stomach discomfort in animals. Rats were intolerant to the gluten food. Gluten food in rat creates constipation. In this Food-related model of FGID, 2 Wistar rats were divided into 4 groups. The first group was taken as the control group (Normal Saline), the second group was taken as the standard group (bisacodyl 20mg/kg), the third group was taken as Test I (proteases 500mg/kg) and the fourth group was taken as Test II (proteases 1000mg/kg). All rats were placed in a cage individually (one rat in each cage). After that animals were given gluten food (bread) as a diet instead of a normal diet. This procedure was followed for 14 days and every day the faecal droppings of rats were counted. However, gluten food causes constipation in rats. After completion of 14 days, the rats were administered with standard and test drugs and checked faecal count for 24 hours¹³. There is brain-gut interaction in FGID. So, we also check the brain serotonin level of rats.

Antidiarrheal activity (Castor-oil induced model):

Although there are many symptoms of FGID such as acidity, bloating, and constipation. Diarrhoea is one of the important symptoms of FGID is related to the gut-brain. In this model, the animals were divided into 4 groups having 6 animals in each. Group I was treated as the control group (normal saline), group II was treated as the control group (Loperamide 2mg/kg), group III was treated as test I (lower dose of protease 500mg/kg) and group IV was treated as test II (higher dose of protease 1000mg/kg). After giving respective drug and saline to each group, the animals in each group were received 1 ml castor oil orally after 60 minutes of medication treatment, also watery faeces and the number of defecations were measured for up to 4 hours in the clear metabolic cages with filter paper at the base¹⁴.

Water avoidance stress model:

The severity of the FGID symptoms predicted by daily chronic stress, according to several studies. These discoveries led to the development of rodent models with altering stress exposure, and water avoidance was identified as most potent psychological stressors having this feature. Data from the literature revealed that prolonged administration of water avoidance stress for ten days in rats- a strain high responsiveness- provoked colonic hyperalgesia, increased or decreased colonic motility etc.¹⁵

In water avoidance stress rats produced GI problems. In water avoidance stress model, the test equipment consisted of a plastic tank with a block positioned in the middle of the floor, measuring 45 centimetre in length, 25 centimetre in width and 25 centimetre in height. Fresh normal water was poured into tank until it was 1 centimetre from the top of block. The rats were placed on the block for one hour each day for ten days straight as part of the chronic stress regimen. In water avoidance stress model, the animals distributed to four groups – Group I was treated as control group, Group II was treated as standard group (Bisacodyl 20mg/kg), Group III was treated as test I (lower dose of protease 500 mg/kg) and Group IV was treated as test II (higher dose of protease 1000mg/kg). This standard and test drug was given after 10 days of procedure and after that measured the change in faecal count and level of brain serotonin^16-17.

Elevated Plus Maze Model:

The CNS activity was also measured using Swiss albino mice in order to correlate with the previously documented potential of protease on GI-disorder with the brain (ENS-CNS) correlation in common terms FGID’s. This experiment was performed because most accepted theory of FGID is there is link between gut and brain.

The Elevated Plus Maze was an animal model of anxiety. Two tight arms 50*10*40 CM as well as two open arms 50*10 CM was composed of black-colored hardwood planks and was joined to a central platform 10*10 CM. Both open and closed arms were faced one other and were surrounded by a removable cover. The maze was elevated to fifty cm. above the ground. This approach is rapid, accurate and appropriate for determining the effects of anxiolytic medications under normal conditions. All animals were distributed to 4 groups in which six mice in each. Group I was considered as control, and they were given saline. The second group as Group II was functioned as control group and received diazepam (1mg/kg), The test groups, Group III and IV received proteases at dose of 500 mg/kg as well as 1000mg/kg respectively. Diazepam administered I.P. and all other doses were administered orally. All treatments were administered 30 minutes before taking the observations¹⁸.

Estimation of Serotonin:

After being anaesthetized with isoflurane, 48 rats from all groups of the food-related model of FGID and the water avoidance stress paradigm were carefully dissected. Brain tissue was homogenized in a Teflon tissue homogenizer at 0ºC. The homogenates were centrifuged at 14000 R.P.M. for 15minutes at 0 degree C after being placed on ice. Crystal clear supernatant solutions were combined with 0.5ml of NaOH (1N) and 1ml of zinc sulphate (10%). Before centrifuging the tubes for 20minutes at 2500rpm, they were shaken for five minutes. 1ml of crystal-clear supernatant solution was combined with 0.3ml (2Normal of HCl) in quartz cuvette. The intensity of fluorescence in resultant solution was determined using a fluorimeter (SYSTONIC fluorimeters S-9150) at wavelength [excitation (nanometer)/ fluorescence (nanometer)] 290/550¹⁹.

RESULT:

Acute oral toxicity study:

The proteases which were isolated from the latex of T. divaricata was tested for acute oral toxicity using the OECD guideline no 423. The investigation was conducted using the mature Swiss albino mice weighing 25-30 gm in good health. Four groups of three animals each were formed, and the animals fasted for one night. The doses for groups I, II, III, and IV were 5, 50, 300, and 2000mg/kg respectively. Several measures including body temperature, deification, etc., were measured after protease administration. After administration of protease, the toxicity was checked up to 14 days but there was no toxicity on a higher dose of 2000mg/kg. So below 2000mg/kg dose will be considered as effective dose. After completing oral toxicity study, one lower dose 500mg/kg, and one higher dose of protease 1000mg/kg were selected for further pharmacological screening after formulation.

Evaluation of NPLT formulation:

Physical Appearance: The formulated nanoemulgels were observed to be clear, and transparent with good consistency. It showed a with the polymer for proper application.

pH: the pH of the nanoemulgels was observed to be between 5.5 to 7.0 which is perfect for their intranasal administration as its measured pH is near to the pH of the nasal mucosa. The formulated nanogels will be non-irritant to the nasal mucosa. With easy application.

Spreadability: The spreadability of the nanoemulgels was found to be in a range of 8.5 gcm/s to 10.3 gcm/s which was ideal for their best application intranasally.

Evaluation of NPLT for Functional gastrointestinal disorder (FGID):

Food-related model of FGID:

Constipation was remained in the control group of the food related mode of FGID. It was discovered in the standard group that faecal count increased but turned normal within a short interval of time. It was discovered in Oral administration of Protease that the faecal count was increasing which means that the test drug is treating the FGID induced constipation. The effect of 1000 mg/kg (p<0.0001) dose of Protease’s effect was much more effective in comparison to lower dose of protease which was 500mg/kg. In this model I also checked brain serotonin level of rat because of brain-gut interaction.

Anti-diarrheal model (Castor oil induced):

When diarrhoea was induced in this model then it was observed that control group had more diarrhoea count and delay in the defecation time was less. Contrary to this, when treated with standard drug (Loperamide) diarrhoeal faecal count reduced much earlier and defecation time increased. When animals were treated with NPLT containing Protease (500mg/kg and 1000 mg/kg) then decrement in the diarrhoeal count was seen and there was also change in defecation time. NPLT with 1000mg/kg (p<0.0001) higher dose of protease was more effective than 500mg/kg.

Table 1: Effect of protease on faecal count

Group	Cumulative Number of faecal at different time interval
Group	After 2 hours	After 4 hours	After 6 hours	After 8 hours	After 10 hours
Control	0.500±0.224	1.000±0.000	1.500±0.224	2.000±0.000	2.500±0.224
Standard (Bisacodyl) (20mg/kg)	7.833±0.601	16.333±0.882	25.833±1.014	33.500±0.992	36.000±0.365
NPLT – 500	6.167±0.601****	12.500±0.764****	20.167±11.470***	24.833±0.910****	27.000±1.065****
NPLT - 1000	7.333±0.667****	14.667±0.715****	24.167±0.749****	30.167±0.601****	33.833±0.477****

With n = 6 in each group, the data was reported as mean ± SEM. In statistical analysis, ANOVA was used. At lower and higher content (500 mg and 1000 mg), the significance of test I found to be (***p<0.0001 at 6 hours and ****p<0.0001 for other time interval) and for test II was found to be (****p<0.0001) while comparing it with control.

Table 2: Effect of proteases on castor oil induced diarrhoea

Treatment	No. of diarrhoeal faecal	Defecation time (min)
Control	8.833±0.6009	3.5±0.2236
Standard (Loperamide)	4.5±0.4282	7.0±0.5774
NPLT - 500	7.500±0.6191**	5.167±0.3073*
NPLT – 1000	5.167±0.3073*	6.0±0.3651**

With n = 6 in each group, the data was reported as mean ± SEM. In statistical analysis, ANOVA was used. For no. of diarrheal faecal at lower and higher content, the significance of test I found to be (**p<0.005) and for test II was found to be (*p<0.01) and for defecation time test I and test II found to be *p<0.01 and **p<0.005 respectively while comparing it with control.

Estimation of serotonin:

In the suggested study, the serotonin level in control group decreased and was found to be less than the average serotonin. In comparison to control rat, the rats treated with both dosages of proteases showed a substantial increment in serotonin level. Proteases had a more effective impact on brain serotonin levels at 1000 mg/kg.

Table 5: Level of serotonin in rat brain

Groups	Level of serotonin
Groups	Food related model of FGID	Water avoidance stress model
Control	69.50±1.5	70.33±1.647
Standard (Bisacodyl 20mg/kg)	115.7±1.256	117.2±2.857
NPLT – 500	96.67±1.430****	86.17±1.167**
NPLT – 1000	103.50±1.996***	99.83±0.9458****

With n = 6 in each group, the data was reported as mean ± SEM. In statistical analysis, ANOVA was used. In food related model, at lower and higher dose (500mg/kg and 1000 mg/kg), the significance of test I found to be ****p<0.0001 and for test II was found to be ***p<0.0001, and in WAS model, at lower dose the significance for test I found to be **p<0.001 and for test II found to be **** p<0.0001in comparison to control.

Effect of proteases on iliac motility:

The proteases from T. divaricata exhibited contraction on iliac motility in Water avoidance stress and food related model of FGID that result enhancement of gut iliac motility and the percentage response was found 100% at dose of 500 µg/m and 700 µg/ml respectively.

Fig. 1: Effect of protease on iliac motility. The graph showed proteases contract the iliac on both dose (lower and higher). This indicates proteases had effect on gut motility.

DISCUSSION:

The term FGID refers to chronic and recurrent gastrointestinal irregularities and symptoms. The term “Gut-brain connection disorders,” or FGID, refers to a group of complex illnesses that affect several digestive tract organs and have symptoms such gut hypersensitivity and motility problem. The gut-brain axis is a bidirectional pathway via which changed gut physiological and psychological variables interact, FGD is the clinical syndrome of this pathway. Throughout the evolution of FGIDs, the interaction between the Gastrointestinal brain (ENS) and the CNS is crucial. Even while FD and IBS are very well-known FGIDs, many other conditions are growing in popularity, including constipation and abdominal pain syndrome and these are functional.

According to the results of the current study, psychosocial factors may have a role in the progression and development of IBD as well as how patients manage these debilitating and chronic conditions. According to several specialists, IBD patients frequently have CNS issues such as anxiety and sadness. According to research, anxiety and grief are quite prevalent, with up to 37% of resolution and 83% of anxiety during recurrence.

Today, several synthetic medications, including corticosteroids, immunosuppressants, and aminosalicylates are often recommended to treat FGIDs. In most situations, they have therapeutic potential, but they are also frequently accompanied by adverse effects on patients’ quality of life and compliance. Patients receiving long-term treatment with these medications exhibited substantial life problems. To prevent such consequences, it is necessary to look for an alternative and complementary therapy or to utilize a herbal medicine.

Food indigestion, intolerance, or allergy are common causes of abdominal discomfort. Oral tolerance, which is crucial in preventing immunological reactivity against food antigens, may be altered due to genetic predisposition, illness, or stress, leading to FGID symptoms. Certain food molecules cause FGID symptoms in IBS patients, and elimination diet techniques, such as gluten-free diets, are useful in some individuals.

Nanoemulgels are scientifically proven and observed to be effective for gastrointestinal diseases by acting at the site of action GIT and the brain stem after their oral administration^19-20. In the present study the prepared nanoemulgels of the isolated herbal protease enhanced the bioavailability along with targeted effect of the medication.

The result of present research showed that protease which is obtained from latex of T. divaricata is effective for FGIDs symptoms. To decide the dose of proteases we have conducted Oral toxicity study. After these studies were conducted, the 4 models of FGIDs which is the food-related model of FGID, Anti-diarrhoeal activity (castor oil-induced), the Water avoidance stress model, and the elevated plus maze anxiety model were taken and I evaluated the effect of proteases.

In the food-related model, the normal diet was replaced with a gluten-containing diet (bread) which induced constipation in rats. After the oral administration the faecal count increases indicating the proteases are affected against constipation. The higher dose of proteases showed more potent results at lower and higher doses (500 mg/kg and 1000 mg/kg), and the significance of the test I found to be (***p<0.0001 at 6 hours and ****p<0.0001 for another time interval) and for test II was found to be (****p<0.0001) while comparing it with control.

On the other hand, the Water avoidance stress model is anxiety anxiety-induced FGID model. In this model, the rats were kept at a 10 cm long platform and that platform was placed in the water tank which is filled with water. This led to induced anxiety in rats and ultimately caused constipation. After those animals treated with proteases and proteases showed significant results by increment in faecal count. This normalization of faecal count indicates proteases is effective against this model. At lower and higher doses (500mg/kg and 1000 mg/kg), the significance of test I was found to be (p<0.001 at 2 hours and ****p<0.0001 for other time intervals) and for test II was found to be (****p<0.0001) while comparing it with control.

Diarrhoea is the main symptom of FGIDs. To check Anti-diarrhoeal activity, castor oil is used to induce diarrhoea in rats. After induction of diarrhoea, the rats were treated with proteases with lower and higher doses. Protease cures diarrhoea by decreasing the diarrhoeal faecal count and increasing the faecal count. The proteases are performed by mainly managing the faecal output. the significance of test I was found to be (**p<0.005) and of test II was found to be (*p<0.01) and for defecation time test I and test II were found to be *p<0.05 and **p<0.001 respectively while comparing it with control while comparing it with control.

EPM model is chosen for FGID because FGID is a disorder of brain-gut interaction and EPM is a model for anxiety which is directly linked to gut interaction. In normal conditions when mice were placed to EPM, the mice spent most of the time in closed arms and entries are also rare in closed arms due to the anxiety. When animals are treated with proteases orally there are changes in their activity. Now mice spend more time in the open and entries to arms also increase. This showed that proteases also have an anxiolytic effect. At lower and higher dose (500mg/kg and 1000 mg/kg), the significance of test I on no. of entries in open arms was found to be **p<0.001 and for test II was found to be **p<0.005. At lower and higher doses, the significance of test I and test II on time spent in open and closed arms was found to be ****p<0.0001 while comparing it with control.

Any fluctuation in serotonin level causes problems in the gastrointestinal. When the serotonin levels were checked in 2 models which were food-related models and then found that the control group which was treated with only saline solution had a lower level than the normal serotonin level and the proteases treated showed the normal level of serotonin. So basically, proteases work by normalizing the serotonin level in the brain In the food-related model, at the lower and higher doses (500mg/kg and 1000 mg/kg), the significance of the test I found to be ****p<0.0001 and for test II was found to be ***p<0.0001, and in WAS model, at the lower dose the significance for the test I found to be **p<0.001 and for test II found to be ****p<0.0001 in comparison to control.

The iliac motility was checked for 2 models and found that the 100 % response of iliac motility for water avoidance stress was at 2.77 log and dose and for the food-related model was at log dose 2.84. The proteases exhibited contraction in iliac motility in Food related stress model of FGID that resulted from the the enhancement of iliac motility and the percentage response was found 100% at the dose of 1000 µg/ml in water avoidance stress model the percentage response was found 100 % at the dose of 500 µg/ml.

From the above discussions, it is found that nanoemulgel of proteases are highly effective against symptoms of FGIDs like diarrhoea, constipation, bloating etc. and protease lowering frequency of these symptoms effectively.

CONCLUSION:

Functional Gastrointestinal Disorder is associated with chronic gastrointestinal issues and can’t be addressed by pathological conditions. Based on the pathophysiology of FGID which is still unclear and the major symptoms of the targeted disease, the present study investigated the novel and targeted formulation nanoemulgel of the protease from T. divaricate; NPLT at the oral administration of 500 mg/kg and 1000 mg/kg against it. It can also be confirmed that the process behind proteases screening action against FGIDs is its capacity to increase serotonin levels in brain. It also involved evaluation of iliac motility and found out that NPLT provided contractive effect on iliac tissue. The overall study demonstrated the role of NPLT in the management of FGID by normalizing the faecal count and by reducing the anxiety. Proteases may have several mechanisms to treat FGID included brain serotonin level. The work as a contributes to the world of medicine by offering a fresh and effective anti-FGID medicine.

As a potential treatment alternative of FGIDs, NPLT is thought to be a strong anti-FGIDs agent. The current study can serve as a foundation for future research into potent alternative components derived from readily accessible latex from the abundant plant T. divaricate making it more economic. Protease, which is proteolytic enzyme found in animals, plant. Proteases have many therapeutic properties like wound-healing, anti-ulcer etc. In FGIDs proteases play an important role. Proteases cure FGIDs symptoms like constipation, diarrhoea etc. Proteases also normalized the brain serotonin level which is the primary cause of FGIDs.

ACKNOWLEDGEMENT:

We acknowledge Noida Institute of Engineering and Technology, Greater Noida for allowing us to conduct this research work.

CONFLICT OF INTEREST:

There is not any conflict of interest between the authors as it is a teamwork.

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Received on 08.02.2025 Revised on 05.09.2025

Accepted on 03.01.2026 Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):2909-2916.

DOI: 10.52711/0974-360X.2026.00415

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Group	Cumulative faecal counts at different time interval
Group	After 2 hours	After 4 hours	After 6 hours	After 8 hours	After 10 hours
Control	1.000±0.258	1.333 ± 0.211	1.833 ±0.307	2.167±0.307	2.333±0.211
Standard (Bisacodyl (20 mg/kg)	7.167±0.307	13.333±0.558	21.333±0.422	28.333±0.760	31.167±1.302
NPLT-500	4.833±0.401***	12.167±0.601****	17.500±0.563****	23.167±0.477****	27.167±0.601****
NPLT-1000	5.833±0.307****	12.833±0.601****	18.667±0.667****	24.833±0.749****	29.000±0.931****

Name of Groups	No. of entries in arms (n)		Time spent in arms (n)
Name of Groups	Open arms	Closed arms	Open arms	Closed arms
Control	6.5±0.342	11.833±0.307	37.83±0.601	185.66±1.909
Standard (diazepam1mg/kg)	14.5±0.764	3.167±0.477	86.83±0.872	104.83±4.175
NPLT- 500	7.833±0.307**	7.5±0.428**	57.167±1.4****	152.83±1.537****
NPLT - 1000	10.833±0.601**	5.833±0.307***	75.667±1.87****	124.167±1.014****